Peptides for the treatment of Alzheimer&#39;s disease and other beta-amyloid protein fibrillogenesis disorders

ABSTRACT

A pharmaceutical composition comprising peptide HA3G76 Tyr-Leu-Ser-Lys-Gly-Arg-Leu-Val-Phe-Ala-Leu-Gly (SEQ ID NO:8).

This application is a division of U.S. patent application Ser. No.09/962,955 filed Sep. 24, 2001 now U.S. Pat. No. 6,933,280, which is acontinuation-in-part of U.S. patent application Ser. No. 09/938,275filed Aug. 22, 2001, which is a continuation of U.S. patent applicationSer. No. 08/947,057 filed Oct. 8, 1997 now abandoned which claimedpriority to U.S. Provisional Application 60/027,981 filed Oct. 8, 1996.

This invention was made with government support under 1 R43 AG17787-01awarded by the National Institute on Aging. The Government has certainrights in the invention.

TECHNICAL FIELD

This invention relates to the use of laminin peptides and lamininderivatives for the treatment of Alzheimer's disease and otherbeta-amyloid protein fibrillogenesis disorders. This invention alsorelates to the use of laminin peptides and laminin derivatives asamyloid-fibril forming agents and compounds that are able to enhance Aβfibrillogenesis.

BACKGROUND OF THE INVENTION

Background for therapeutic use of laminin and peptide fragments oflaminin in the treatment of Alzheimer's disease and other amyloidosescan be found in U.S. patent application Ser. No. 09/938,275 filed Aug.22, 2001, the text and drawings of which are hereby incorporated byreference into the present application as if fully set forth herein.

Beta-Amyloid Protein as a Therapeutic Target for Alzheimer's Disease

Alzheimer's disease (AD) is characterized by the deposition andaccumulation of a 39–43 amino acid peptide termed the beta-amyloidprotein, Aβ or β/A4 (Glenner and Wong, Biochem. Biophys. Res. Comm.120:885–890. 1984; Masters et al, Proc. Nat. Acad. Sci. U.S.A.82:4245–4249, 1985; Husby et al, Bull. WHO 71:105–108,1993). Aβ isderived from larger precursor proteins termed beta amyloid precursorproteins (or APPs) of which there are several alternatively splicedvariants. The most abundant forms of the APPs include proteinsconsisting of 695, 751 and 770 amino acids (Kitaguchi et al, Nature331:530–532, 1988; Ponte et al, Nature 331:525–527,1988; Tanzi et al,Nature 331:528–530, 1988). The small Aβ peptide is a major componentthat makes up the core of amyloid deposits called “plaques” in thebrains of patients with AD. In addition, AD is characterized by thepresence of numerous neurofibrillary “tangles”, consisting of pairedhelical filaments which abnormally accumulate in the neuronal cytoplasm(Grundke-Iqbal et al Proc. Natl. Acad. Sci. U.S.A. 83:4913–4917., 1986;Kosik et al, Proc. Natl. Acad. Sci. U.S.A. 83:4044–4048, 1986; Lee etal, Science 251:675–678, 1991). The other major type of lesion found inAD brain is the accumulation of amyloid in the walls of blood vessels,both within the brain parenchyma and meningeal vessels that lie outsidethe brain. The amyloid deposits localized to the walls of blood vesselsare referred to as cerebrovascular amyloid or congophilic angiopathy(Mandybur, J. Neuropath. Exp. Neurol. 45:79–90, 1986; Pardridge et al,J. Neurochem. 49:1394–1401, 1987). The pathological hallmarks of ADtherefore are the presence of “plaques”, “tangles”, and cerebrovascularamyloid deposits.

For many years there has been an ongoing scientific debate as to theimportance of “amyloid” in AD and whether the “plaques” and “tangles”characteristic of this disease, were a cause or merely the consequencesof the disease. Recent studies indicate that amyloid is indeed acausative factor for AD and should not be regarded merely as aconsequence. The Alzheimer's Aβ protein in cell culture has been shownto cause degeneration of nerve cells within a short time period (Pike etal, Br. Res. 563:311–314, 1991; J. Neurochem. 64:253–265, 1995). Studiessuggest that it is the fibrillar structure, characteristic of allamyloids, that is mainly responsible for the neurologic effects. Aβ hasalso been found to be neurologic in slice cultures of hippocampus(Hadrian et al, Neurobiol. Aging 16:779–789, 1995) and induces nervecell death in transgenic mice (Games et al, Nature 373:523–527, 1995;Hsiao et al, Science 274:99–102, 1996). Injection of Aβ into rat brainalso causes memory impairment and neuronal dysfunction (Flood et al,Proc. Natl. Acad. Sci. U.S.A. 88:3363–3366, 1991; Br. Res. 663:271–276,1994). Convincing evidence that Aβ amyloid is directly involved in thepathogenesis of AD comes from genetic studies. It was discovered thatthe increased production of Aβ could result from mutations in the geneencoding, its precursor, APP (Van Broeckhoven et al, Science248:1120–1122, 1990; Murrell et al, Science 254:97–99, 1991; Haass etal, Nature Med. 1:1291–1296, 1995). The identification of mutations inthe APP gene which causes early onset familial AD is a strong argumentthat Aβ and amyloid are central to the pathogenetic process underlyingthis disease. Four reported disease-causing mutations have now beendiscovered which demonstrate the importance of Aβ in causing familial AD(reviewed in Hardy, Nature Gen. 1:233–234, 1992). Lastly, recent studiessuggest that a reduction in amyloid plaque load in APP transgenic micelead to improvements in behavioral impairment and memory loss (Chen etal, Nature 408:978–982, 2000; Janus et al, Nature 408:979–982, 2000;Morgan et al, Nature 408:982–985, 2000). This is the strongest argumentto date that implicates that reduction of Aβ amyloid load in brainshould be a central target for the development of new and effectivetreatments of AD and related disorders.

Alzheimer's Disease and the Aging Population

Alzheimer's disease is a leading cause of dementia in the elderly,affecting 5–10% of the population over the age of 65 years (Jorm, AGuide to Understanding of Alzheimer's Disease and Related Disorders, NewYork University Press, New York, 1987). In AD, the parts of the brainessential for cognitive processes such as memory, attention, language,and reasoning degenerate. In some inherited forms of AD, onset is inmiddle age, but more commonly, symptoms appear from the mid-60's onward.AD today affects 4–5 million Americans, with slightly more than half ofthese people receiving care in many different health care institutions.The prevalence of AD and other dementias doubles every 5 years beyondthe age of 65, and recent studies indicate that nearly 50% of all peopleage 85 and older have symptoms of AD (NIH Progress Report on AD,National Institute on Aging, 2000). Thirty-three million people of thetotal population of the United States are age 65 and older, and thiswill climb to 51 million people by the year 2025 (NIH Progress Report onAD, National Institute on Aging, 2000). The annual economic toll of ADin the United States in terms of health care expenses and lost wages ofboth patients and their caregivers is estimated at $80 to $100 billion(NIH Progress Report on AD, National Institute on Aging, 2000).

Tacrine hydrochloride (“Cognex”), the first FDA approved drug for AD isan acetylcholinesterase inhibitor (Cutler and Sramek, N. Engl. J. Med.328:808–810, 1993). However, this drug has showed limited success in thecognitive improvement in AD patients and initially had major sideeffects such as liver toxicity. The second and third FDA approved drugsfor AD, are donepezil (“Aricept”)(Barner and Gray, Ann. Pharmacotherapy32:70–77, 1998; Rogers and Friedhoff, Eur. Neuropsych. 8:67–75, 1998),and rivastigmine tartrate (“E2020” or “Exelon”) (Polinsky, Clin. Ther.20:634–647, 1998; Ballard and McAllister, Pychopharmacol. 146:10–18,1999), which are also acetylcholinesterase inhibitors and more effectivethan Tacrine in demonstrating slight cognitive improvements in ADpatients, but are not believed to be a cure. Therefore, it is clear thatthere is a need for more effective treatments for AD patients. In thepresent invention, we have identified laminin globular domain-derivedpeptides that serve as potent inhibitors of Aβ fibril formation andgrowth, and which cause disruption/disassembly of preformed AD fibrils.

Laminin and Its Presence in Alzheimer's Disease

Laminin is a large glycoprotein complex of 850 kDa which normallyresides on the basement membrane and is produced by a variety of cellsincluding embryonic, epithelial and tumor cells (Foidart et al Lab.Invest. 42:336–342, 1980; Timpl, Eur. J. Biochem. 180:487–502, 1989).Laminin interacts with various extracellular matrix components includingheparan sulfate proteoglycans (Riopelle and Dow, Br. Res. 525:92–100,1990; Battaglia et al, Eur. J. Biochem. 208:359–366, 1992), heparin(Sakashita et al, FEBS Letts. 116:243–246, 1980; Del-Rosso et al,Biochem. J. 199:699–704, 1981; Skubitz et al, J. Biol. Chem.263:4861–4868, 1988) and type IV collagen (Terranova et al, Cell22:719–726, 1980; Rao et al, Biochem. Biophys. Res. Comm. 128:45–52,1985; Charonis et al, J. Cell Biol. 100:1848–1853, 1985; Laurie et al,J. Mol. Biol. 189:205–216, 1986). Laminin is composed of three distinctpolypeptide chains, A1, B1 and B2 (also referred to as alpha-1, β1 andgamma-1, respectively), joined in a multidomain cruciform structurepossessing three short arms and one long arm (Burgeson et al, MatrixBiol. 14:209–211, 1994). Studies involving in vitro self-assembly andthe analysis of cell-formed basement membranes have shown that the threeshort arms interact to form a polymer which is a part of a basementmembrane network (Yurchenco et al, J. Biol. Chem. 260:7636–7644, 1985;J. Cell Biol. 117:1119–1133, 1992; Yurchenco and Cheng, J. Biol. Chem.268:17286–17299, 1993). In addition to its role in basement membraneformation (Kleinman et al, Biochem. 22:4969–4974, 1983), laminin alsoplays important roles in a number of fundamental biological processesincluding promotion of neurite outgrowth (Lander et al, Proc. Natl.Acad. Sci. U.S.A. 82:2183–2187, 1985; Bronner-Fraser and Lallier, J.Cell Biol. 106:1321–1329, 1988) and cell adhesion (Engvall et al, J.Cell Biol. 103:2457–2465, 1986). Injury to adult brain also induceslaminin production by astrocytes (Liesi et al, EMBO J. 3:683–686, 1984)indicating its role in repair processes. In AD and Down's syndrome,laminin is believed to be present in the vicinity of Aβ amyloid plaques(Perlmutter and Chui, Br. Res. Bull. 24:677–686, 1990; Murtomaki et al,J. Neurosc. Res. 32:261–273, 1992; Perlmutter et al, Micro. Res. Tech.28:204–215, 1994). Previous studies have also indicated that the variousisoforms of APP of AD bind laminin (Narindrasorasak et al, Lab. Invest.67:643–652, 1992) and other basement membrane components, includingperlecan (Narindrasorasak et al, J. Biol. Chem. 266:12878–12883, 1991),fibronectin and type IV collagen (Narindrasorasak et al, J. Biol. Chem.270:20583–20590, 1995).

DISCLOSURE OF THE INVENTION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/938, 275 filed Aug. 22, 2001, the contents of which arehereby incorporated by reference into the present application as iffully set forth herein.

Methods are disclosed herein for the treatment and diagnosis ofAlzheimer's disease and other disorders that involve the accumulationand persistence of beta-amyloid protein (Aβ). Methods are disclosed fortreating Alzheimer's disease and other Aβ disorders, comprisingadministering to a subject or patient a therapeutically effective doseof at least one laminin globular domain-derived peptide, or an analog ora derivative thereof. In one exemplary embodiment, the laminin peptidewhich is a potent Aβ amyloid inhibitory agent is selected from the groupconsisting of AG73 (SEQ ID NO:1), C-16 (SEQ ID NO:2), A-13 (SEQ IDNO:3), HA3G47 (SEQ ID NO:4), HA3G58 (SEQ ID NO:5), HA3G67 (SEQ ID NO:6),HA3G74 (SEQ ID NO:7), HA3G76 (SEQ ID NO:8), HA3G79 (SEQ ID NO:9), HA3G83(SEQ ID NO:10), A4G82 (SEQ ID NO:11), A5G15 (SEQ ID NO:12), A5G56 (SEQID NO:13), A5G80 (SEQ ID NO:14), A5G81 (SEQ ID NO:15), A5G82 (SEQ ID NO:16), A5G84 (SEQ ID NO:17), A5G101 (SEQ ID NO:18), A5G109 (SEQ ID NO:19),hereinafter referred to for easy reference as Sequence Group A, but morepreferably selected from the group consisting of AG73 (SEQ ID NO:1),A-13 (SEQ ID NO:3), HA3G76 (SEQ ID NO:8), A4G82 (SEQ ID NO:11), A5G81(SEQ ID NO: 15) and A5G101 (SEQ ID NO:18), hereinafter referred to foreasy reference as Sequence Group B.

In addition, methods are disclosed for the use of specific lamininglobular domain-derived peptides, or an analog or a derivative thereof,not for the inhibition of amyloid fibrils, but for the formation ofamyloid-like fibrils or as amyloid enhancing agents, for instance as anaid for diagnostics and in vitro testing, the better to judge theefficacy of the disclosed inhibitory compounds. In one exemplaryembodiment, the laminin peptide which is an Aβ amyloid enhancing agentis selected from the group consisting of A-13 (SEQ ID NO:3), HA3G47 (SEQID NO:4), HA3G58 (SEQ ID NO:5), HA3G83 (SEQ ID NO:10), LAM-L (SEQ IDNO:20), A4G10 SEQ ID NO:21), A4G46 (SEQ ID NO:22), A4G47 (SEQ ID NO:23),A4G84 (SEQ ID NO:24), A4G92 (SEQ ID NO:25), A4G107 (SEQ ID NO:26), A5G3(SEQ ID NO:27), A5G10 (SEQ ID NO:28), A5G27 (SEQ ID NO:29), A5G33 (SEQID NO:30), A5G65 (SEQ ID NO:31), A5G77 (SEQ ID NO:32), A5G87 (SEQ IDNO:33), A5G90 (SEQ ID NO:34) and A5G111 (SEQ ID NO:35), hereinafterreferred to for easy reference as Sequence Group C.

The laminin peptides of the present invention may be prepared by knownchemical synthetic methods or by biotechnological methods. Assays usefulfor the screening and identification of laminin peptide analogs asinhibitors of Aβ fibrillogenesis are also disclosed. In addition,methods are disclosed for the labeling of polypeptides derived from theinvention for diagnosis of Alzheimer's and other Aβ amyloidoses.

The present invention relates to the novel and surprising discovery thatlaminin globular-domain derived peptides are inhibitors of Alzheimer'sdisease amyloidosis, and therefore have potential use for thetherapeutic intervention of Alzheimer's disease and related Aβdisorders.

It is therefore an object of the present invention is to provide amethod for treating Alzheimer's disease and other disorders involvingthe formation and persistence of Aβ, comprising the administration oflaminin-derived peptides.

Another object of the present invention is to disclose specific lamininglobular domain-derived peptides and other novel analogs and derivativesthereof, the administration of which comprises a method for treatingAlzheimer's disease and other Aβ amyloidoses.

The invention also relates to pharmaceutical compositions comprising thelaminin globular domain-derived peptides and other analogs andderivatives of such peptides, or pharmaceutically acceptable saltsthereof for use in the treatment of Alzheimer's disease and other Aβamyloidoses.

As used herein the term “laminin globular domain-derived peptide” isused to include each laminin globular domain-derived peptide which wassurprisingly found to inhibit Aβ fibrillogenesis as disclosed herein,analogs, derivative and fragments thereof that retain the activity ofthe complex peptide. The term analogs are intended to include variantson the peptide molecule brought about, for example, homologoussubstitution of individual or several amino acid residues. The termderivative is used to include minor chemical changes that may be made toeach of the laminin globular domain-derived peptides themselves oranalogs thereof that maintain the biological activity of each of theparent peptides disclosed.

The invention also discloses the identity of several laminin globulardomain-derived peptides that have the ability to form amyloid-likefibrils themselves and therefore serve as amyloid enhancing agents.

The invention also discloses methods to utilize the laminin-derivedpeptides as diagnostic or imaging agents for Alzheimer's disease andother Aβ amyloidoses.

The invention also discloses methods to utilize antibodies made againstlaminin-derived peptides as therapeutic agents for the treatment ofAlzheimer's disease and other Aβ amyloid disorders.

A primary object of the present invention is to establish newtherapeutic methods for Alzheimer's disease and other disease involvingthe accumulation of Aβ. These Aβ diseases include, but are not limitedto, the amyloid associated with Alzheimer's disease and Down's syndrome,and various forms of cerebral amyloidosis, known to those knowledgeablein the art.

A primary object of the present invention is to use laminin globulardomain derived peptides as potent inhibitors of Aβ amyloid formation,deposition, accumulation and/or persistence in Alzheimer's disease andother Aβ amyloidoses. Laminin globular domain derived peptides include,but are not limited to, the peptides of Sequence Group A, and morepreferably the peptides of Sequence Group B and/or A5G109 (SEQ IDNO:19).

Yet another object of the present invention is to use analogs orderivatives thereof of each of the laminin globular domain derivedpeptides as potent inhibitors of Aβ amyloid formation, deposition,accumulation and/or persistence in Alzheimer's disease and other Aβamyloidoses. Laminin globular domain derived peptides include but arenot limited to, the peptides of Sequence Group A, and more preferablythe peptides of Sequence Group B.

Yet another object of the present invention is to use peptidomimeticcompounds modeled from the laminin globular domain peptides disclosedherein, including but not limited to, the peptides of Sequence Group A.

Yet another aspect of the present invention is to make use of lamininglobular domain-derived peptides including, but not limited to, thepeptides of Sequence Group A, and fragments or analogs thereof, aspotential therapeutics to inhibit the deposition, formation andaccumulation of fibrillar amyloid in Alzheimer's disease and other Aβamyloidosis disorders, and to enhance the clearance and/or removal ofpre-formed amyloid deposits in brain (for Alzheimer's disease and Down'ssyndrome and other Aβ amyloidoses).

Yet another object of the present invention is to use the lamininglobular domain-derived peptides of the present invention, and allconstituents, analogs or variants thereof, including peptides which haveat least 70% identity to the sequences disclosed herein. Specificlaminin globular domain-derived peptides as described above may bederived from any species including, but are not limited to, human,murine, bovine, porcine, and/or equine species.

Yet another object of the present invention is to use laminin globulardomain-derived peptides as described herein as a specific indicator forthe presence and extent of laminin breakdown in brain by monitoringbiological fluids including, but not limited to, cerebrospinal fluid,blood, serum, urine, saliva, sputum and stool.

Yet another object of the present invention is to make use of peptidesor analogs or derivatives thereof as described herein, including but notlimited to, the peptides of Sequence Group A, as potential blockingtherapeutics for the interaction of laminin and laminin-derivedfragments in a number of biological processes and diseases (such as inAlzheimer's disease, Down's syndrome and other amyloid diseases).

Another object of the present invention is to use pills, tablets,caplets, soft and hard gelatin capsules, lozenges, sachets, cachets,vegicaps, liquid drops, elixers, suspensions, emulsions, solutions,syrups, tea bags, aerosols (as a solid or in a liquid medium),suppositories, sterile injectable solutions, and sterile packagedpowders, which contain laminin globular domain-derived peptides,including but not limited to, the peptides of Sequence Group A, andanalogs, derivatives or fragments thereof, to treat patients withAlzheimer's disease and other Aβ amyloidoses.

Yet another object of the present invention is to provide compositionsand methods involving administering to a subject a therapeutic dose oflaminin globular domain-derived peptides, which inhibit Aβ amyloiddeposition, including but not limited to, the peptides of Sequence GroupA, and analogs, derivatives or fragments thereof. Accordingly, thecompositions and methods of the invention are useful for inhibitingamyloidosis in disorders in which amyloid deposition occurs. Thepeptides of the invention can be used therapeutically to treatamyloidosis or can be used prophylactically in a subject susceptible toamyloidosis. The methods of the invention are based, at least in part,in directly inhibiting Aβ amyloid fibril formation, and/or causingdissolution of pre-formed Aβ amyloid fibrils.

Yet another object of the present invention is to provide pharmaceuticalcompositions for treating Aβ amyloidosis. The pharmaceuticalcompositions include a therapeutic compound of the invention in anamount effective to inhibit Aβ amyloid deposition and a pharmaceuticallyacceptable vehicle.

Yet another object of the present invention is to use laminin globulardomain-derived peptides as amyloid agents or amyloid enhancing agents,including but not limited to, the peptides of Sequence Group C.

Yet a further aspect of the present invention is to use anti-idiotypicantibodies to laminin-derived protein fragments and/or laminin-derivedpolypeptides as potent inhibitors of amyloid formation, deposition,accumulation and/or persistence in Alzheimer's disease and other Aβamyloidoses.

Another aspect of the invention is to provide new and novel polyclonaland/or monoclonal peptide antibodies which can be utilized in a numberof in vitro assays to specifically detect Aβ-binding laminin derivedprotein fragments and/or Aβ-binding laminin derived polypeptides inhuman tissues and/or biological fluids. Polyclonal or monoclonalantibodies that are made specifically against a peptide portion orfragment of laminin which interacts with Aβ can be utilized to detectand quantify amyloid disease specific laminin fragments in human tissuesand/or biological fluids. These antibodies can be made by administeringthe peptides in antigenic form to a suitable host. Polyclonal ormonoclonal antibodies may be prepared by standard techniques known tothose skilled in the art.

Another object of the present invention is to use laminin-derivedpolypeptides referred to above, for the detection and specificlocalization of laminin peptides important in the amyloid diseases inhuman tissues, cells, and/or cell culture using standardimmunohistochemical techniques.

Yet another aspect of the present invention is to use antibodiesrecognizing any of the Aβ-binding laminin fragments, and/orlaminin-derived polypeptides including, but not limited to, the peptidesof Sequence Group A, and analogs, derivatives or fragments thereof, forin vivo labeling; for example, with a radionucleotide, for radioimagingto be utilized for in vivo diagnosis, and/or for in vitro diagnosis.

Another object of the present invention is to use Aβ-bindinglaminin-derived polypeptides or fragments thereof, in conjunction withpolyclonal and/or monoclonal antibodies generated against these peptidefragments, using in vitro assays to detect amyloid disease specificautoantibodies in human biological fluids. Specific assay systems can beutilized to not only detect the presence of autoantibodies againstAβ-binding laminin-derived protein fragments or polypeptides thereof inbiological fluids, but also to monitor the progression of disease byfollowing elevation or diminution of laminin protein fragments and/orlaminin-derived polypeptide autoantibody levels.

Another aspect of the invention is to utilize laminin-derived proteinfragments and/or laminin-derived polypeptide antibodies and/or molecularbiology probes for the detection of these laminin derivatives in humantissues in the amyloid diseases.

Yet another object of the present invention is to use thelaminin-derived protein fragments or polypeptides of the presentinvention in each of the various therapeutic and diagnostic applicationsdescribed above. The laminin-derived protein fragments include, but arenot limited to, a ˜55 kDa fragment of laminin generated by trypsindigestion, a ˜55 kDa fragment of laminin generated by elastasedigestion, and a ˜30 kDa fragment of laminin generated by trypsindigestion. The laminin-derived polypeptides include, but are not limitedto the peptides of Sequence Group A, and analogs, derivatives orfragments thereof, including peptides which have at least 70% identityto the sequences disclosed herein. Specific laminin-derived proteinfragments or peptides as described above may be derived from any speciesincluding, but are not limited to, human, murine, bovine, porcine,and/or equine species.

Another object of the invention is to provide polyclonal and/ormonoclonal peptide antibodies that can be utilized in a number of invitro assays to specifically detect laminin protein fragments orpolypeptides in human tissues and/or biological fluids. Polyclonal ormonoclonal antibodies made specifically against a peptide portion orfragment of any of the laminin fragments or polypeptides describedherein can be utilized to detect and quantify laminin-derived proteinfragments or laminin-derived polypeptides in human tissues and/orbiological fluids. These antibodies can be made by isolating andadministering the laminin-derived fragments and/or polypeptides inantigenic form to a suitable host. Polyclonal or monoclonal antibodiesmay be prepared by standard techniques by one skilled in the art.

Yet another object of the present invention is to use laminin-derivedfragment or polypeptide-derived antibodies as described herein as aspecific indicator for the presence and extent of laminin breakdown inbrain by monitoring biological fluids including, but not limited to,cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.

Yet another object of the present invention is to use laminin-derivedfragment or polypeptide antibodies as described herein as a specificindicator for the presence, extent and/or progression of Alzheimer'sdisease and/or other brain amyloidoses by monitoring biological fluidsincluding, but not limited to, cerebrospinal fluid, blood, serum, urine,saliva, sputum, and stool.

Yet another object of the present invention is to use laminin-derivedfragment or polypeptide-derived antibodies as described herein as aspecific indicator for the presence and extent of laminin breakdown insystemic organs by monitoring biological fluids including, but notlimited to, cerebrospinal fluid, blood, serum, urine, saliva, sputum,and stool.

Yet another object of the present invention is to use laminin-derivedfragment or polypeptide antibodies as described herein as a specificindicator for the presence and extent of amyloidosis in type II diabetesby monitoring biological fluids including, but not limited to,cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.

Yet another object of the present invention is to use laminin-derivedfragment or polypeptide antibodies as described herein as a specificindicator for the presence and extent of amyloidosis in other systemicamyloidoses by monitoring biological fluids including, but not limitedto, cerebrospinal fluid, blood, serum, urine, saliva, sputum, and stool.

Yet another object of the present invention is to make use of peptidesor fragments of laminin as described herein, including but not limitedto, the peptides of Sequence Group A, and fragments thereof, aspotential blocking therapeutics for the interaction of laminin andlaminin-derived fragments in a number of biological processes anddiseases (such as in Alzheimer's disease and other amyloid diseasesdescribed herein).

Yet another object of the invention is to utilize specificlaminin-derived fragment or polypeptide antibodies, as described herein,for the detection of these laminin fragments in human tissues in theamyloid diseases.

Preferred pharmaceutical compositions have at least one laminin peptideor fragment thereof selected from the group consisting of AG73 (SEQ IDNO:1), C-16 (SEQ ID NO:2), A-13 (SEQ ID NO:3), HA3G47 (SEQ ID NO:4),HA3G58 (SEQ ID NO:5), HA3G67 (SEQ ID NO:6), HA3G74 (SEQ ID NO:7), HA3G76(SEQ ID NO:8), HA3G79 (SEQ ID NO:9), HA3G83 (SEQ ID NO:10), A4G82 (SEQID NO:11), A5G15 (SEQ ID NO:12), A5G56 (SEQ ID NO:13), A5G80 (SEQ IDNO:14), A5G81 (SEQ ID NO:15), A5G82 (SEQ ID NO: 16), A5G84 (SEQ IDNO:17), A5G101 (SEQ ID NO:18) (also together referred to herein asSequence Group A) and A5G109 (SEQ ID NO:19).

In preferred embodiments the composition have the structureArg-Lys-Arg-Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg-Thr (SEQ ID NO: 1) orArg-Gln-Val-Phe-Gln-Val-Ala-Tyr Ile-Ile-Ile-Lys-Ala (SEQ ID NO:3) orTyr-Leu-Ser-Lys-Gly-Arg-Leu-Val-Phe-Ala-Leu-Gly (SEQ ID NO:8) orThr-Leu-Phe-Leu-Ala-His-Gly-Arg-Leu-Val-Phe-Met (SEQ ID NO:11) orAla-Gly-Gln-Trp-His-Arg-Val-Ser-Val-Arg-Trp-Gly (SEQ ID NO:15) orAsp-Gly-Arg-Trp-His-Arg-Val-Ala-Val-Ile-Met-Gly (SEQ ID NO:18).

In any of the above structures or sequences, the individual amino acidsmay be either L- or D-amino acids. The pharmaceutical composition have atherapeutically effective amount of any of the above structures orsequences, preferably together with a pharmaceutically acceptablecarrier, diluent or excipient.

Preferred pharmaceutical agents for treating Aβ amyloidosis in a patienthave a therapeutically effective amount of a polypeptide selected fromSequence Group A or A5G109 (SEQ ID NO:19), and have an Aβ amyloidinhibitory activity or efficacy greater than 30%, as compared to dulyestablished controls, such as patients who do not received the preferredpharmaceutical agent.

An important Aβ amyloidosis to which the disclosed therapeutics areaddressed is Alzheimer's disease. A preferred therapeutically effectamount of disclosed polypeptide is a dosage in the range of from about10 μg to about 50 mg/kg body weight/per day, and more preferably in therange of from about 100 μg to about 10 mg/kg body weight per day.

The pharmaceutical agent may advantageously be administered in aparenterally injectable or infusible form or orally.

A method is also disclosed to diagnose a disease or susceptibility to Aβamyloidosis related to the level of laminin-derived polypeptides. Firstthe levels of laminin-derived polypeptides in a sample are determined,whereby the levels are indicative of the presence of Aβ amyloidosis,susceptibility to Aβ amyloidosis, or progression of Aβ amyloidosis. Inpreferred methods the laminin-derived polypeptides are selected from thegroup consisting of Sequence Group A and/or A5G109 (SEQ ID NO:19).

The sample assayed may be a biological fluid, and the biological fluidmay be serum derived from humans.

A method of making an antibody is also disclosed, the method producingantibodies from a peptide sequence selected from the group consisting ofSequence Group A and/or A5G109 (SEQ ID NO:19), and fragments thereof.The method preferably includes production of at least one type ofantibody selected from the group consisting of polyclonal, monoclonal,chimeric, and anti-idiotypic antibodies and monitoring a biologicalfluid for the presence and extent of laminin-derived polypeptides as anindicator for the extent of an amyloid disease and radiolabeling theantibodies for radioimaging or in vivo diagnosis for detection oflaminin-derived protein fragments or laminin-derived polypeptides.

A method of forming amyloid-plaque like deposits in vitro is alsodisclosed. The method includes incubating a laminin-derived polypeptideat 37° C. for 3 to 7 days and selecting the laminin-derived polypeptidefrom the group consisting of LAM-L (SEQ ID NO:20), A-13 (SEQ ID NO:3),HA3G47 (SEQ ID NO:4), HA3G58 (SEQ ID NO:5), HA3G83 (SEQ ID NO:10), A4G10SEQ ID NO:21), A4G46 (SEQ ID NO:22), A4G47 (SEQ ID NO:23), A4G84 (SEQ IDNO:24), A4G92 (SEQ ID NO:25), A4G107 (SEQ ID NO:26), A5G3 (SEQ IDNO:27), A5G10 (SEQ ID NO:28), A5G27 (SEQ ID NO:29), A5G33 (SEQ IDNO:30), A5G65 (SEQ ID NO:31), A5G77 (SEQ ID NO:32), A5G87 (SEQ IDNO:33), A5G90 (SEQ ID NO:34) and A5G111 (SEQ ID NO:35) (also togetherreferred to herein as Sequence Group A). These steps also advantageouslydefine an alternate method for enhancing Aβ amyloid fibril formation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention.

FIG. 1 is a graph demonstrating an inhibitory effect of Aβ amyloiddeposition into rodent hippocampus by laminin.

FIG. 2 is a copy of a black and white photograph of a Coomassie bluestained gel demonstrating purification and isolation of fragments oflaminin which strongly interact with Aβ.

FIG. 3 is a graph demonstrating the strong binding interaction ofAlzheimer's Aβ to the ˜55 kilodalton laminin fragment. A singledissociation constant with a K_(d)=2.0×10⁻⁹ was determined.

FIG. 4 is a graph demonstrating the inhibition of Alzheimer's Aβ fibrilformation by selected fragments disclosed herein.

FIG. 5 is a schematic representation of the sequence of human alpha-3chain (SEQ ID NO: 36) globular domain peptides disclosed herein (SEQ IDNOS 40–88 disclosed respectively in order of appearance).

FIG. 6 is a schematic representation of the sequence of murine alpha-4chain (SEQ ID NO: 37) globular domain peptides disclosed herein (SEQ IDNOS 89–97, 21, 98–132, 22–23, 133–168, 24, 169–175, 25, 176–189, 26 and190–198, disclosed respectively in order of appearance).

FIG. 7 is a schematic representation of the sequence of murine alpha-5chain (SEQ ID NO: 38) globular domain peptides disclosed herein (SEQ IDNOS 199–200, 27, 201–206, 28, 207–222, 29, 223–227, 30, 228–258, 31,259–269, 32, 270–278, 33, 279–280, 34, 281–300, 35 and 301–302).

FIG. 8 is a table which includes laminin globular domain-derivedpeptides which can disrupt/disassemble pre-formed Alzheimer's Aβ1–40fibrils (SEQ ID NOS 1–20, 118,229,240,264, 300 and 303–307).

FIG. 9 is a graph demonstrating further testing of selected lamininglobular-domain derived peptides against pre-formed Alzheimer's Aβ 1–42fibrils.

FIG. 10 is a graph demonstrating dose-dependent disruption/disassemblyof pre-formed Aβ 1–42 fibrils by laminin globular domain-derivedpeptides.

FIG. 11 is a composite color photograph demonstrating amyloid enhancingeffects of laminin-derived peptides.

BEST MODE OF CARRYING OUT THE INVENTION

Accordingly the use of laminin-derived peptides for the treatment ofAlzheimer's disease and other Aβ amyloidoses is disclosed. Specificallyobserved and isolated laminin globular domain-derived peptides disclosedherein have the ability to inhibit Aβ fibril formation, and cause adisruption of pre-formed Aβ amyloid fibrils, and therefore possesstherapeutic potential in the treatment of Alzheimer's disease and otherdisorders involving the formation, deposition, accumulation andpersistence of Aβ.

Pharmaceutically acceptable salts of the peptides disclosed in thepresent invention include both salts of the carboxy groups and the acidaddition salts of the amino groups of the peptide molecule. Salts of thecarboxy groups may be formed by methods known in the art and includeinorganic salts such as sodium, calcium ammonium, ferric or zinc saltsand the like and salts with organic bases such as those formed withamines such as triethanolamine, arginine or lysine, piperidine, procaineand the like. Acid addition salts include salts with mineral acids suchas hydrochloric acid and sulphuric acid and salts of organic acids suchas acetic acid or oxalic acid.

The pharmaceutical composition may contain laminin-derived peptides suchas those disclosed herein as unique peptides or in polymerized orconjugated form attached to macromolecular carriers or polymers. Thecompositions may optionally contain pharmaceutically acceptableexcipients. In an alternative embodiment the composition may contain thelaminin-derived peptide alone.

The route of administration includes oral, intravenous,intra-peritoneal, intra-muscular, subcutaneous, intra-articular,intra-nasal, intra-thecal, intra-dermal, transdermal or by inhalation.An effective dose of each of the laminin-derived peptides disclosedherein as potential therapeutics for use in treating Aβ amyloidosis inAlzheimer's disease and other disorders be from about 1 μg to 500 mg/kgbody weight, per single administration, which may readily be determinedby one skilled in the art. The dosage depends upon the age, sex, health,and weight of the recipient, kind of concurrent therapy, if any, andfrequency of treatment.

As used herein the laminin-derived polypeptides of the present inventionmay consist of -L amino acid, -D amino acids or a mixture of both forms.Amino acids in nature usually consist of -L amino acids. However,substitution with -D amino acids may demonstrate enhanced Aβ amyloidinhibitory activity, enhanced bioavailability due to less degradation inbiological fluids (such as plasma), and enhanced penetration across theblood-brain-barrier. Polypeptides having an identical amino acidsequence to that found within a parent peptide but which all or part ofthe L-amino acids have been substituted with D-amino acids is part ofthe present invention for the development of therapeutics to treatAlzheimer's disease and other Aβ amyloidoses.

The -L or -D amino acids of the laminin-derived polypeptides of thepresent invention are further intended to include other peptidemodifications, including derivatives, analogs and mimetics, that retainthe ability of the polypeptides to inhibit Aβ amyloidosis as describedherein. The terms “analog”, “derivative” and “mimetic” as used hereinare intended to include molecules which mimic the chemical structure ofa L or D-peptidic structure, and retain the functional properties of aL- or D-peptidic structure. Approaches to designing peptide analogs,derivatives and mimetics are known in the art. For example, see P. S.Farmer, in Drug Design, E. J. Ariens, ed., Academic Press, New York,1980, v. 10, pp. 119–143; Ball and Alewood, J. Mol. Recognition 3:55,1990; Morgan and Gainor, Ann. Rep. Med. Chem. 24:243, 1989; andFreidinger, Trends Pharmacol. Sci. 10:270, 1989. See also Sawyer,“Peptidomimetic design and chemical approaches to peptide metabolism”,in M D Taylor and G L Amidon, eds., in Peptide-Based Drug Design:Controlling Transport and Metabolism, Ch. 17, 1995; Smith et al, J. Am.Chem. Soc. 117:11113–11123, 1995; Smith et al, J. Am. Chem. Soc.116:9947–9962, 1994; and Hirschman et al, J. Am. Chem. Soc.115:12550–12568, 1993.

As used herein, a “derivative” of a therapeutic compound (e.g. a peptideor polypeptide) refers to a form of the peptide in which one or morereaction groups of the compound have been derivatized with a substituentgroup. Examples of peptide derivatives include peptides in which anamino acid side chain, the peptide backbone, or the amino- orcarboxy-terminus has been derivatized (e.g., peptidic compounds withmethylated amide linkages). As used herein an analog of a therapeuticcompound refers to a compound which retains chemical structuresnecessary for functional activity (i.e. Aβ inhibitory activity), yetwhich also contains certain chemical structures which differ from theparent peptide. An example of an analog of a naturally occurring peptideis a peptide which includes one or more non-naturally-occurring aminoacids. As used herein, a “mimetic” of a compound refers to a compound inwhich chemical structures of the compound are necessary for functionalactivity have been replaced with other chemical structures which mimicthe conformation of the compound or peptides thereof. Examples ofpeptidomimetics include peptide compounds in which the peptide backboneis substituted with one or more benzodiazepine molecules (see James etal, Science 260:1937–1942, 1993).

Analogs of the polypeptide compounds of the invention are intended toinclude compounds in which one or more L- or -D amino acids of thepeptide structure are substituted with a homologous amino acid such thatthe properties of the original polypeptide are maintained. Preferablyconservative amino acid substitutions are made at one or more amino acidresidues. A “conservative amino acid substitution” in one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art, including basic side chains (e.g.lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagines,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Non-limiting examples ofhomologous substitutions that can be made in the peptidic structures ofthe invention include substitution of phenylalanine with tyrosine,leucine with valine, or other natural or non-natural amino acid havingan aliphatic side chain and/or substitution of valine with leucine orother natural or non-natural amino acid having an aliphatic side chain.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Preferably, the carrier is suitable foradministration into the central nervous system (e.g. intraspinally orintracerebrally). Alternatively, the carrier can be suitable forintravenous, intraperitoneal or intramuscular administration. In anotherembodiment, the carrier is suitable for oral administration.Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is compatible with theactive compound, use thereof in the pharmaceutical compositions of theinvention is contemplated. Supplementary active compounds can also beincorporated into the compositions.

As used here in “Aβ amyloidoses” refers to amyloid diseases whichinvolve the formation, deposition, accumulation and/or persistence of Aβ(i.e. beta-amyloid protein), including but not limited to Aβ containing39–43 amino acids in length, but more preferably, Aβ 1–40 (SEQ IDNO:36), or Aβ 1–42 (SEQ ID NO:37), and mixtures or fragments thereof.

“Aβ amyloidoses” and “Aβ fibrillogenesis diseases” include, but are notlimited to Alzheimer's disease, Down's syndrome, forms of familialamyloidosis, cerebrovascular amyloidosis and cerebral hemorrhage,cystatin C amyloid angiopathy, hereditary cerebral hemorrhage withamyloidosis (Dutch type), hereditary cerebral hemorrhage withamyloidosis (Icelandic type), and inclusion body myositis.

These and other features and advantages of the present invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying figures whichare illustrative of embodiments of the invention only, and are not meantto limit the scope of the invention.

FIG. 1 is a graph demonstrating inhibition of fibrillar Aβ amyloiddeposition into rodent hippocampus by laminin. Laminin caused asignificant 90% inhibition of Aβ amyloid deposition in brain.

FIG. 2 is a black and white photograph of a Coomassie blue stained geldemonstrating purification and isolation of a ˜55 kilodalton fragment oflaminin, and a ˜30 kilodalton subfragment of laminin, identified asfragments of laminin which strongly interact with Aβ.

FIG. 3 is a graph demonstrating the strong binding interaction ofAlzheimer's Aβ to the ˜55 kilodalton laminin fragment. A singledissociation constant with a K_(d)=2.0×10⁻⁹ was determined.

FIG. 4 is a graph demonstrating the inhibition of Alzheimer's Aβ fibrilformation by various protease-generated laminin fragments. Intactlaminin, ˜55 kilodalton and ˜30 kilodalton laminin-fragments obtained bytrypsin digestion, and a ˜55 kilodalton fragment of laminin obtained byelastase digestion, all significantly inhibited Alzheimer's Aβ fibrilformation at 3 and 7 days.

FIG. 5 is a schematic that demonstrates the sequence of peptides derivedfrom the human alpha-3 chain globular domain that was used for screeningfor Alzheimer's Aβ amyloid inhibitory activity. A total of 49 12–14amino acid peptides (labeled A3G40 to A3G88) were synthesized and usedfor screening studies.

FIG. 6 is a schematic that demonstrates the sequence of peptides derivedfrom the mouse alpha-4 chain globular domain that was used for screeningfor Alzheimer's Aβ amyloid inhibitory activity. A total of 117 12–14amino acid peptides (labeled A4G-1 to A4G-116) were synthesized and usedfor screening studies.

FIG. 7 is a schematic that demonstrates the sequence of peptides derivedfrom the mouse alpha-5 chain globular domain that was used for screeningfor Alzheimer's Aβ amyloid inhibitory activity. A total of 133 12–14amino acid peptides (labeled A5G-1 to A5G-113) were synthesized and usedfor screening studies.

FIG. 8 is a table that identifies the laminin globular domain-derivedpeptides which can disrupt/disassemble pre-formed Alzheimer's Aβ 1–40fibrils.

FIG. 9 is a graph demonstrating the further testing of selected lamininglobular-domain derived peptides against pre-formed Alzheimer's Aβ 1–42fibrils.

FIG. 10 is a graph demonstrating dose-dependent disruption/disassemblyof pre-formed Aβ 1–42 fibrils by laminin globular domain-derivedpeptides.

FIG. 11 are color photographs which demonstrate the amyloid enhancingeffects of laminin-derived peptides. Figure A demonstrates the formationof congophilic maltese-cross amyloid plaque-like deposits (arrows)formed by the laminin-derived peptide A5G3 (SEQ ID NO:27). Figure Bdemonstrates the formation of congophilic maltese-cross amyloidplaque-like deposits (arrows) formed by the laminin-derived peptideAG510 (SEQ ID NO:28). Figure C demonstrates congophilic amyloid deposits(arrows) (but no maltese-cross amyloid plaque-like formation) of Aβ 1–42(SEQ ID NO: 37) alone. Figure D demonstrates congophilic amyloiddeposits (but no maltese-cross amyloid plaque-like formation) of Aβ 1–42(SEQ ID NO:37) alone. Figure E demonstrates the formation of congophilicmaltese-cross amyloid plaque-like deposits formed following theco-incubation of Aβ 1–42 (SEQ ID NO:37) and laminin-derived peptide A5G3(SEQ ID NO:27). Figure F demonstrates the formation of congophilicmaltese-cross amyloid plaque-like deposits (arrows) formed following theco-incubation of Aβ 1–42 (SEQ ID NO:37) and laminin-derived peptideA4G107 (SEQ ID NO:26). Figures A–D are at a magnification of 200×,whereas Figures E and F are at a magnification of 400×.

EXAMPLES

The following examples are provided to disclose in detail preferredembodiments of the potent inhibitory effects of laminin fragments, andlaminin globular domain-derived peptides on Aβ fibrillogenesis. However,it should not be construed that the invention is limited to thesespecific examples.

Example 1 Inhibition of Fibrillar Aβ Amyloid Deposition in RodentHippocampus by Laminin

The effects of laminin on Aβ amyloid deposition in brain was evaluatedusing a rodent model (Snow et al, Neuron 12:219–234, 1994). In thisstudy, 50 μg Aβ 1–42 (Bachem, Torrance, Calif., U.S.A.), or 50 μg Aβ1–42+50 μg laminin (Sigma Chemical Co., St. Louis, Mo., U.S.A.; EHStumor), were infused (using Alzet osmotic pumps) directly intohippocampus for 1 week in adult Sprague-Dawley rats (250–300 grams; 3months old; n=8 per group). In addition, to assess the effects oflaminin on neuronal cell viability and integrity, a group of animals wasinfused with only 50 μg of laminin. Infusion of Aβ only into hippocampusfor 1 week using Alzet osmotic pumps produced amyloid deposits thatstained extensively with Congo red (and demonstrated a red/greenbirefringence when viewed under polarized light). In contrast,co-infusion of Aβ+laminin prevented deposition of fibrillar Aβ amyloid,as shown by a reduction in the red/green birefringence and congophiliaof the amyloid deposit. Examination of brain tissues from groups ofanimals infused with laminin alone demonstrated no neuronal loss, andcomplete neuronal integrity in brain tissue. This latter observationsuggested that laminin had little effect in altering normal brainarchitecture. The extent of amyloid deposition was assessed by blindscoring (by 2 investigators) of Congo red stained sections (as viewedunder polarized light) throughout the infusion site using an arbitraryscale as previously described (Snow et al, Neuron 12:219–234, 1994).These studies conclusively demonstrated that laminin inhibited Aβ fibrildeposition in brain. Blind scoring of Congo red stained brain sectionsrevealed that the group infused with Aβ only, had a Congo red score of2.0+/−0.54 (mean±SD), consistent with previous studies (Snow et al,Neuron 12:219–234, 1994)(FIG. 1). In the presence of laminin, asignificant 90% inhibition (p<0.01) of Aβ amyloid deposition wasobserved (FIG. 1).

Example 2 Purification of a ˜55 kDa Fragment and ˜30-kDa Sub-Fragment ofLaminin that Binds Aβ

To generate enough of the ˜55 kDa laminin-fragment, and its ˜30 kDasub-fragment, 10 mg of EHS mouse laminin (Sigma Chemical Co., St. Louis,Mo., U.S.A.) was digested with elastase as described in Example 7 (ofU.S. patent application Ser. No. 08/947,057; filed on Oct. 7, 1997 andhereby incorporated as reference herein) and purified byelectrophoresis. Briefly, intact EHS laminin was left undigested, ordigested with elastase (Sigma Chemical Co., St. Louis, Mo., U.S.A.)prior to SDS-PAGE. More specifically, 0.1 mg of elastase in 200 μl of 50mM Tris-HCl buffer (pH 8.0) were added to 5 ml of laminin (10 mg) in thesame buffer and incubated at 37° C. for 2.5 hr, and a 5 μl aliquot wastaken for analysis, whereas the remainder was immediately frozen at −80°C. These conditions have been worked out for optimal generation of the˜55 kDa laminin fragment, and the generation of the ˜55 kDa lamininfragment was usually confirmed by electrophoresis of a 5 μl aliquot. Themixture of fragments was separated in 10% polyacrylaminde gelpreparative electrophoresis using a Model 491 Prep Cell (BioRad,) with aRediFrac fraction collector (Pharmacia). A portion of the purified ˜55kDa fragment was further digested with trypsin under the sameconditions, with peptide to trypsin weight ratios similar to theconditions used for elastase digestion (as described above). A ˜30 kDasub-fragment (of the ˜55 kDa protein) was purified in a similar manner(using the Model 491 Prep Cell) as described above.

FIG. 2 demonstrates a Coomassie blue stained gel of a trypsin digest oflaminin (lane 1), the purified ˜30 kDa laminin sub-fragment producedfollowing trypsin digestion of laminin (lane 2), the purified ˜55 kDalaminin-fragment following elastase digestion of laminin (lanes 3 and4), and the purified ˜30 kDa product following trypsin digestion of the˜55 kDa laminin-derived protein of lane 4 (lane 5). Molecular weightstandards are shown in the far left lane.

Example 3 Isolated Laminin Globular Domains Bind Aβ (1–40) with a SingleAffinity

Since the staining of the ˜55 kDa fragment of laminin by ligand blotanalysis using Aβ 1–40 as a probe was so dramatic (not shown), wehypothesized that the ˜55 kDa fragment of laminin (which containsprimarily laminin globular domains) must bind very tightly to Aβ. Tostudy the interaction of Aβ (1–40) with the ˜55 kDa laminin fragment, asolid phase binding immunoassay was used, whereby the isolated ˜55 kDalaminin fragment was immobilized on microtiter plates and incubated withincreasing concentrations of Aβ (1–40). Aβ 1–40 was found to bind the˜55 kDa laminin-fragment in a concentration dependent and saturablefashion, with an apparent single dissociation constant of K_(d)=2.0×10⁻⁹M (FIG. 3). When the amount of Aβ (1–40) bound to the wells wasdecreased, the K_(d) values obtained were identical, indicating anaccurate K_(d) determination (Engel and Schalch, Mol. Immunol.17:675–680, 1980; Fox et al, EMBO J. 10:3137–3146, 1991; Castillo et al,J. Neurochem. 69:2452–2465, 1997; Mann et al, Eur. J. Biochem.178:71–80, 1988). The high affinity of the ˜55 kDa laminin-fragment toAβ and its potent anti-Aβ amyloid activity (see below) indicated thatthis laminin fragment contained regions that interacted with Aβ quitewell.

Example 4 Inhibition of Aβ 1–40 Fibril Formation by Protease-GeneratedLaminin Fragments

To determine whether the laminin fragments generated by trypsin andelastase were capable of inhibiting Aβ amyloid fibril formation, theability of laminin-derived fragments (believed to represent portions ofthe globular domains of laminin) to inhibit Aβ 1–40 fibril formationover a 1 week period was tested. For this study purified proteinsincluding, intact laminin, the ˜55 kDa and ˜30 kDa fragments of lamininobtained by trypsin digestion, and the ˜55 kDa fragment of lamininobtained by elastase digestion, were incubated with 25 μM Aβ 1–40(Bachem, Torrance, Calif., U.S.A.) at 1:1 weight ratios (equivalent toAβ:laminin fragment molar ratios of 200:1, 13:1, 7:1, and 13:1,respectively). As shown in FIG. 4, 25 μM of freshly solubilized Aβ(1–40) when incubated alone at 37° C. gradually increased influorescence from 1 hour to 1 week. One hundred twenty-five nM oflaminin significantly (p<0.05) inhibited Aβ fibril formation at 3 daysand 7 days, in agreement with previous studies. Similarly, the purified˜55 kDa and 30 kDa fragments of laminin were also found to significantly(p<0.05) inhibit Aβ fibril formation in a similar manner, at 3 days and1 week of incubation. The ˜55 kDa laminin fragment obtained by elastasedigestion (which was confirmed by amino acid microsequencing torepresent the C-terminal globular domains of the laminin A1 chain) alsoinhibited Aβ 1–40 fibril formation similar to that observed with the ˜55kDa laminin fragment obtained by trypsin digestion. These studies alsosuggested that both ˜55 kDa laminin fragments generated by eithertrypsin or elastase digestion, likely contained similar if notidentical, peptide regions which caused inhibition of Aβ fibrilformation. This study confirmed that the globular domain regions oflaminin contains Aβ amyloid inhibitory sequences, and justified the nextset of studies which involved the screening of hundreds of synthesizedpeptides (12–14 amino acids in length) which represented overlappingregions within the globular domain of different laminin chains.

Example 5 Screening of Peptides from the Globular Domain Regions ofDifferent Laminin Chains

Our initial studies demonstrated that the globular domain region oflaminin was involved in binding to Aβ, and in the inhibition of Aβfibril formation (see U.S. patent application Ser. No. 08/947,057). Inthe next set of studies, a series of overlapping 12–14 amino acidpeptides against the globular domain regions of the alpha-1, alpha-3(FIG. 5), alpha-4 (FIG. 6) and alpha-5 (FIG. 7) chain of laminin weresynthesized. More than 300 (12–14 amino acid) peptides corresponding tothe globular domain regions of the various laminin chains weresynthesized manually using the 9-fluorenylmethoxy-carbonyl (FMOC) methodand C-terminal amides. The respective amino acids were condensedmanually in a stepwise manner using 4-(2″,4″-dimethoxyphenyl-FMOC-amino-methyl)-phenoxy resin (Rink, TetrahedronLett. 28:3787–3790, 1987). The amino acid side chain protecting groupswere removed as described previously (Nomizu et al, J. Biol. Chem.269:30386–30392,1994; J. Biol. Chem. 270: 20583–20590, 1995). Theresulting protected synthetic peptide resins were de-protected andcleaved from the resins using trifluoroacetic acid-thianisole-m-cresolethanedithiol-H₂O (80:5:5:5:5) at 20° C. for 3 hours. Crude peptideswere then precipitated and washed with ethyl ether and purified byreverse phase HPLC using a Vydac 5C18 column with a gradient ofwater/acetonitrile containing 0.1% trifluoroacetic acid. The purity ofthe peptides was confirmed by analytical HPLC. The identity of eachpeptide was confirmed using a Sciex API IIIE triple quadruple ion spraymass spectrometer (Otaka et al J. Org. Chem. 60:3967–3974, 1995). Morethan 300 peptides were synthesized for Aβ amyloid inhibitory activityscreening using Thioflavin T fluorometry (Castillo et al J. Neurochem.69:2452–2465, 2000). For initial screening studies, 25 μM of pre-formedAβ 1–40 fibrils were incubated for 7 days with various 12–14 amino acidlaminin globular domain-derived peptides at an Aβ:peptide molar ratio of1:6. Of 300 peptides screened, only 30 peptides (listed in FIG. 8) werefound to demonstrate a disruption/disassembly greater than 20%. Thesignificance was determined using the paired t-test and comparingfluorescence units ±S.D. (n=3) of Aβ alone versus Aβ+laminin-derivedpeptides.

Example 6 Laminin Globular Domain Peptides that Disrupt/DisassemblePre-formed Aβ Fibrils

FIG. 8 lists 30 laminin globular domain peptides that were able to causea disruption/disassembly of pre-formed Aβ 1–42 fibrils. TheseAlzheimer's amyloid inhibitor peptides included 3 peptides from thelaminin alpha-1 chain globular domain (peptides AG73, LAM-L, and A13;FIG. 8), 1 peptide from the laminin gamma-1 chain (peptide C-16; FIG.8), 11 peptides from the laminin alpha-3 chain globular domain (peptidesA3; HA3G45; HA3G47; HA3G58; HA3G67; HAG371; HAG374; HAG375; HAG376;HAG379, and HAG383; FIG. 8), 2 peptides from the laminin alpha-4 chainglobular domain (peptides A4G31 and A4G82; FIG. 8), and 12 peptides fromthe laminin alpha-5 chain globular domain (peptides A5; A5G15; A5G35;A5G46; A5G46; A5G56; A5G71; A5G80; A5G81; A5G82; A5G84; A5G101; A5G109and A5G110; FIG. 8).

Example 7 Further Testing of Selected Laminin Globular Domain-DerivedPeptides for Aβ 1–42 Amyloid Fibril Inhibitory Activity

From the screening results shown in Example 6 and FIG. 8, we identified19 peptides (out of >300 screened) that were effective in causing a >25%disruption/disassembly of pre-formed Aβ 1–40 fibrils. These includedpeptides AG73 (SEQ ID NO:1), C-16 (SEQ ID NO: 2), A-13 (SEQ ID NO:3),HA3G47 (SEQ ID NO: 4), HA3G58 (SEQ ID NO: 5), HA3G67 (SEQ ID NO: 6),HA3G74 (SEQ ID NO: 7), HA3G76 (SEQ ID NO: 8), HA3G79 (SEQ ID NO: 9),HA3G83 (SEQ ID NO: 10), A4G82 (SEQ ID NO: 11), A5G15 (SEQ ID NO: 12),A5G56 (SEQ ID NO: 13), A5G80 (SEQ ID NO: 14), A5G81 (SEQ ID NO: 15),A5G82 (SEQ ID NO: 16), A5G84 (SEQ ID NO:17), A5G101 (SEQ ID NO:18)(Sequence Group A) and A5G109 (SEQ ID NO: 19).

These selected laminin globular domain-derived peptides were then testedfor their effectiveness to also disrupt/disassemble pre-formed Aβ 1–42fibrils (FIG. 9). In this latter study, selected laminin globulardomain-derived peptides were incubated with pre-formed Aβ 1–42 fibrilsat an Aβ:peptide molar ratio of 1:10. Direct comparisons were made toiAβ5, a 5 amino-acid (LPFFD) Aβ inhibitor previously identified as apotent inhibitor of Aβ fibrillogenesis (Soto et al, Nature Med.4:822–826, 1998) The results demonstrated that six laminin globulardomain-derived peptides were significantly more effective than iAβ5 incausing a disruption/disassembly of preformed Aβ 1–42 fibrils (FIG. 9).These laminin-derived peptides included peptides from the lamininalpha-1 chain [(i.e. AG73—SEQ ID NO:1; A13—SEQ ID NO 3), the lamininalpha-3 chain (i.e. HA3G76—SEQ ID NO:8), the laminin alpha-4 chain (i.e.A4G82—SEQ ID NO: 11) and the laminin alpha-5 chain (i.e. A5G81—SEQ IDNO:15; A5G101—SEQ ID NO: 18). It should be noted that two of these Aβinhibiting peptides were derived from the globular domain of the lamininalpha-1 chain, and the more effective of these two peptides (i.e.AG73—SEQ ID NO:1) was precisely located within the 4th globular domainof the laminin-1 chain, and found to bind very tightly to Aβ (FIG. 8).In our studies (described above), the AG73 (SEQ ID NO:1) peptidedisrupted Ap 1–42 fibrils by 81% when used at an Aβ:peptide molar ratioof 1:10. In comparison, this peptide was 31% more effective than thepreviously described iAβ5 peptide (Soto et al, Nature Med. 4:822–8261998), which in our studies only dissociated pre-formed Aβ 1–42 fibrilsby 50%. At an Aβ:peptide molar ratio of 1:2, the AG73 peptide (SEQ IDNO:1) was also found to disrupt/disassemble pre-formed Aβ 1–42 fibrilsby 72%, whereas the iAβ5 peptide only caused a 27%disruption/disassembly (FIG. 9). The other laminin fragment reported inthe literature (Monji et al, Neurosc. Lett. 251:65–68, 1998) required anAβ:peptide molar ratio of 1:10 to obtain a 50% inhibition of Aβ fibrilformation, whereas our newly identified AG73 peptide (SEQ ID NO:1) onlyrequired an Aβ:peptide molar ratio of 1:1 to achieve the same level ofinhibition. Assuming that the 12-amino acid peptide, AG73 (SEQ ID NO:1),represents a single-site of Aβ binding, we can be confident that we areclose to theoretically optimum inhibition. During this screeningprocess, we also identified 5 other peptides in the alpha 3, 4, and 5chains that were most effective in disrupting/causing a disassembly ofpre-formed Aβ fibrils (see FIG. 8).

Example 8 Dose-Dependent Disassembly of Pre-formed Aβ 1–42 Fibrils byLaminin Globular Domain-Derived Peptides

The next study was implemented to determine whether the six selectedlaminin globular domain-derived peptides were capable of causing adose-dependent disassembly/disruption of pre-formed AD amyloid fibrilscontaining Aβ 1–42. As shown in FIG. 10, disruption of pre-formed ADamyloid fibrils by all six selected laminin-derived peptides occurredfollowing a 7-day incubation period, and in a dose-dependent manner.Significant (p<0.001) disassembly/disruption of pre-formed AD amyloidfibrils containing Aβ 1–42 was observed in the presence of lamininglobular domain-derived peptides and iAβ5. Whereas iAβ5 was effective atall molar ratios tested, the selected laminin peptides were more potent(p<0.05) than iAβ5 at Aβ:peptide molar ratios of 1:2 and 1:10, with thelaminin globular domain derived-peptides showing a range of inhibitionfrom 53–87% compared to a range of inhibition from 27–50% for iAβ5. Atan Aβ:peptide molar ratio of 1:20, the laminin-derived peptides AG73(SEQ ID NO:1), HA3G76 (SEQ ID NO:8), A5G81 (SEQ ID NO:15), A4G82 (SEQ IDNO:11) were all still significantly (p<0.001) more effective than iAβ5.Both A5G101 (SEQ ID NO:18) and A13 (SEQ ID NO:3) have a similareffectiveness to iAβ5 at an Aβ:peptide molar ratio of 1:20. This studytherefore demonstrated that we have identified specific candidatelaminin globular domain-derived peptides that caused adisassembly/disruption of pre-formed AD amyloid fibrils in adose-dependent manner following a 7-day incubation.

Example 9 Identification of Laminin-Derived Peptides that FormAmyloid-like Fibrils

During the screening of >300 laminin globular domain derived peptides,in one study we determined the ability of such peptides to formamyloid-like fibrils. For these studies, following a 1-week incubationat 37° C. of laminin peptides alone, or laminin peptides +25 μM Aβ 1–42,5 μl aliquots of the incubation solutions were placed on gelatin-coatedslides, air-dried over night and stained with Congo red (Puchtler et al,J. Histochem. Cytochem. 10:355–364, 1962). Formation of amyloid-likefibrils was confirmed by the presence of a red/green birefringencefollowing the staining with Congo red, and when viewed under polarizedlight (Puchtler et al, J. Histochem. Cytochem. 10:355–364, 1962. Thefollowing is a list of 20 laminin-derived peptides that have the abilityto form amyloid-like fibrils, as evidenced by a marked red/greenbirefringence following the staining of Congo red and when viewed underpolarized light. These include: LAM-L (alpha-1 chain derived peptide;SEQ ID NO: 20), A-13 (alpha-1 chain derived peptide; SEQ ID NO 3),HA3G47 (alpha-3 chain derived peptide; SEQ ID NO: 4), HA3G58 (alpha-3chain derived peptide; SEQ ID NO: 5), HA3G83 (alpha-3 chain derivedpeptide; SEQ ID NO: 10), A4G10 (alpha-4 chain derived peptide; SEQ IDNO: 21), A4G46 (alpha-4 chain derived peptide; SEQ ID NO: 22), A4G47(alpha-4-chain derived peptide; SEQ ID NO: 23), A4G84 (alpha-4 chainderived peptide; SEQ ID NO: 24), A4G92 (alpha-4 chain derived peptide;SEQ ID NO: 25), A4G107 (alpha-4 chain derived peptide; SEQ ID NO: 26),A5G3 (alpha-5 chain derived peptide; SEQ ID NO: 27)(FIG. 11A), A5G10(alpha-5 chain derived peptide; SEQ ID NO: 28)(FIG. 11B), A5G27 (alpha-5chain derived peptide; SEQ ID NO: 29), A5G33 (alpha-5 chain derivedpeptide; SEQ ID NO: 30), A5G65 (alpha-5 chain derived peptide; SEQ IDNO: 31), A5G77 (alpha-5 chain derived peptide; SEQ ID NO: 32), HA5G87(alpha-5 chain derived peptide; SEQ ID NO: 33), A5G90 (alpha-5 chainderived peptide; SEQ ID NO: 34), and A5G111(alpha-5 chain derivedpeptide; SEQ ID NO: 35) (Sequence Group C). In many instances, thelaminin globular domain derived peptides have the capability to formmaltese-cross congophilic deposits that resemble the maltese-crossamyloid plaques present in Alzheimer's disease brain (FIGS. 11A, 11B).In some instances, the laminin globular-domain derived peptides have theability to enhance Aβ 1–42 peptide to also contain maltese-cross amyloidplaque-like deposits which elicit a red/green birefringence when stainedwith Congo red and viewed under polarized light. These laminin globulardomain derived-peptides that enhance Aβ1–42 plaque-like formationinclude A4G107 (SEQ ID NO:26)(FIG. 11E), A5G3 (SEQ ID NO:27)(FIG. 11F),A5G10 (SEQ ID NO:28), and A5G111 (SEQ ID NO:35)(not shown). FIGS. 11Cand 11D demonstrate the spicule and aggregate Congo red positivedeposits formed by Aβ 1–42 peptide alone.

Example 10 Synthesis of Laminin Globular Domain Analogs

Laminin globular domain-derived peptides (as described above) can beproduced in both the L- and D-amino acid forms. In addition, truncatedpeptides and peptide analogs can be assembled for use as potentialpotent therapeutic peptides for the treatment of Aβ fibrillogenesis inAlzheimer's disease and related disorders. These peptides can beproduced by methods well known to one skilled in the art. For example,L- and D-laminin globular domain-derived peptides could be synthesizedon peptide synthesizers known to those skilled in the art, such as anAdvanced ChemTech Model 396 multiple peptide synthesizer (Louisville,Ky.) using an automated protocol established by the manufacturer for0.025 mmole scale synthesis. Double couplings are performed on allcycles using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU)/N,N-diisopropylethylamine (DIEA)/HOBt/FMOC-AAin four-fold excess for 30 minutes followed by DIC/HOBt/FMOC-AA infourfold excess for 45 minutes. The peptide is then deprotected andremoved from the resin by treatment with TFA/water (95%/5%) for 3 hoursand then precipitated with cold ether. The resulting solid is pelletedby centrifugation (2400 rpm×10 min), and the ether is discarded. Thesolid is then be re-suspended in ether and re-centrifuged for the secondtime after which the ether is decanted for the second time. The solid isdissolved in 10% acetic acid and lyophilized to dryness (˜30 mg for 12amino acid peptides; 18 mg for 7 amino acid peptides). The crude peptideis purified by preparative HPLC using instruments known to those skilledin the art such as a HP 1100 series with diode array detector, with aVydac C18 column (21×250 mm) using a 15%–40% acetonitrile gradient over80 minutes, at a flow rate of 5 ml/min. The primary fraction iscollected and re-analyzed for purity using analytical HPLC to ensure asingle symmetrical peak at all wavelengths. The confirmation ofstructures and sequences is based on comparison of predicted molecularweights to molecular weights obtained by mass spectroscopy. Theseanalyses are performed using instruments known to those skilled in theart, such as a Sciex API IIIE triple quadruple ion spray massspectrometer, for example.

Laminin globular domain derived 12–13 amino acid peptides showing thebest Aβ amyloid inhibitory activity as described in Examples aboveinclude, and are not limited to:

SEQ ID NO: 1  1) AG73 RKRLQVQLSIRT(Arg-Lys-Arg-Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg-Thr) SEQ ID NO: 3  2) A13RQVFQVAYIIIKA (Arg-Gln-Val-Phe-Gln-Val-Ala-Tyr-Ile-Ile-Ile-Lys- Ala) SEQID NO: 8  3) HA3G76 YLSKGRLVFALG(Tyr-Leu-Ser-Lys-Gly-Arg-Leu-Val-Phe-Ala-Leu-Gly) SEQ ID NO: 11 4) A4G82TLFLAHGRLVFM (Thr-Leu-Phe-Leu-Ala-His-Gly-Arg-Leu-Val-Phe-Met) SEQ IDNO: 15 5) A5G81 AGQWHRVSVRWG(Ala-Gly-Gln-Trp-His-Arg-Val-Ser-Val-Arg-Trp-Gly), and SEQ ID NO: 18 6)A5G101 DGRWHRVAVIMG (Asp-Gly-Arg-Trp-His-Arg-Val-Ala-Val-Ile-Met-Gly).

These laminin globular domain-derived peptides can be synthesized usingL- or D-amino acids and can be truncated into shorter 7 or 5 L- orD-amino acid peptides (for example) with or without tyrosine at theC-terminal end.

For example, representative AG73 peptide truncations (the resulting 7 L-or D-amino acid peptides synthesized and tested for amyloid inhibitoryactivity as described below) are RKRLQVQ(Y) (SEQ ID NO: 53), KRLQVQL(Y)(SEQ ID NO: 54), RLQVQLS(Y) (SEQ ID NO: 55), LQVQLSI(Y) (SEQ ID NO: 56),QVQLSIR(Y) (SEQ ID NO: 57) and, VQLSIRT(Y) (SEQ ID NO: 58).

For example, for A13 peptide truncation, a resulting (7 L- or D-aminoacid) peptides synthesized and tested for amyloid inhibitory activity isRQVFQVA (SEQ ID NO: 59), QVFQVAY (SEQ ID NO: 60), VFQVAYI (SEQ ID NO:61), FQVAYII (SEQ ID NO: 62), QVAYIII (SEQ ID NO: 63), VAYIIIK (SEQ IDNO: 64), and AYIIIKA (SEQ ID NO: 65).

For example, for HA3G76 peptide truncation, a resulting (7 L- or D-aminoacid) peptides synthesized and tested for amyloid inhibitory activity isYLSKGRL(Y) (SEQ ID NO: 66), LSKGRLV(Y) (SEQ ID NO: 67), SKGRLVF(Y) (SEQID NO: 68), KGRLVFA(Y) (SEQ ID NO: 69), GRLVFAL(Y) (SEQ ID NO: 70), andRLVFALG(Y) (SEQ ID NO: 71).

For example, for A4G82 peptide truncation, a resulting (7 L- or D-aminoacid) peptides synthesized and tested for amyloid inhibitory activity isTLFLAHG(Y) (SEQ ID NO: 72), LFLAHGR(Y) (SEQ ID NO: 73), FLAHGRL(Y) (SEQID NO: 74), LAHGRLV(Y) (SEQ ID NO: 75), AHGRLVF(Y) (SEQ ID NO: 76), andHGRLVFM(Y) (SEQ ID NO: 77).

For example, for A5G81 peptide truncation, a resulting (7 L- or D-aminoacid) peptides synthesized and tested for amyloid inhibitory activity isAGQWHRV(Y) (SEQ ID NO: 78), GQWHRVS(Y) (SEQ ID NO: 79), QWHRVSV(Y) (SEQID NO: 80), WHRVSVR(Y) (SEQ ID NO: 81), HRVSVRW(Y) (SEQ ID NO: 82), andRVSVRWG(Y) (SEQ ID NO: 83).

For example, for A5G101peptide truncation, a resulting (7 L- or D-aminoacid) peptides synthesized and tested for amyloid inhibitory isDGRWHRV(Y) (SEQ ID NO: 84), GRWHRVA(Y) (SEQ ID NO: 85), RWHRVAV(Y) (SEQID NO: 86), WHRVAVI(Y) (SEQ ID NO: 87), HRVAVIM(Y) (SEQ ID NO: 88), andRVAVIMG(Y) (SEQ ID NO: 89).

Once the above peptides are made, their D-amino acid forms and theirparent L-amino acid forms, along with the truncated 7 L- or D-amino acidpeptides as described above, may advantageously be assayed in vitro foramyloid inhibitory activity as described below. Those that are found tobe efficacious are analyzed further in a number of different in vitroassays such as, to determine their binding affinity to Aβ, their abilityto inhibit Aβ-Aβ self interactions (using a solid phase immunoassay),their effects on disruption/disassembly of α-pleated sheet (usingcircular dichroism spectroscopy), and their ability to inhibit Aβ fibrilformation (by electron microscopy). Peptides that are active are furthertested in cell culture for cellular toxicity, and for their potential toinhibit Aβ-induced neurotoxicity. If incorporation of tyrosine is foundto reduce amyloid inhibitory activity, this step can be stepped by usinga radio labeled D-amino acid as one of the reagents during synthesis toenhance bio-stability.

Active peptides can be linked to polyamine (putrescine, spermidine, orspermine) at the carboxy-terminal ends (Poduslo and Curran, J.Neurochem. 67:734–741, 1996) using the following procedure, as anexample. Briefly, 2 ml of 0.4 M polyamine (putrescine, spermidine, orspermine), pH 4.7 (adjusted with HCl) is used to dissolve 1 mg ofpeptide. To this, 0.2 g of water-soluble1-ethyl-3-(3-dimethylaminopropyl) carbodiamide is added. The reaction isstirred for 4 hours at room temperature and maintained at pH 4.7. Thesolution is purified by preparative HPLC, using instruments known tothose skilled in the art, such as a HP 1100 series with diode arraydetector, with Vydac C18 column (21×250 mm) using 15%-40% acetonitrilegradient over 80 minutes at a flow rate of 5 ml/min. Peptide peaks arepooled and lyophilized for further analysis. Some of the peptides((those containing glutamate (E) or aspartate (D)) have a variablenumber of polyamines attached to them and thus are pooled separatelyfrom those containing one polyamine. The number of polyamines isdetermined based on the molecular weight increase from the parentpeptide as determined, using instruments known to those skilled in theart, such as a Sciex API IIIE triple quadruple ion spray massspectrometer. The polyamine-linked forms of peptides can also be assayedfor amyloid-inhibitory activity (described below). Further truncation ofpeptides can be performed in a similar manner depending on the potencyof the resulting 7 amino acid peptide-analogs. Those that are determinedto be efficacious are synthesized in labeled forms either by iodinationof the tyrosine residues, or during synthesis using radiolabeled aminoacids. The bio-stability of polyamine linkages can also be determinedusing ¹⁴C labeled polyamine available from Sigma (Sigma Chem. Co. StLouis, Mo., U.S.A.).

For the radio-iodination of peptide's tyrosine residues, the followingprocedure is used. Briefly, 0.5 mg of lyophilized peptides in amicrocentrifuge tube is dissolved in 200 μl of 0.5M phosphate buffer (pH7.4), and Na ¹²⁵I solution (2–10 μl; 0.2–1.0 mCi; ICN) is added. Theiodination reaction is initiated by the addition of IodoBeads (Pierce,Rockford, Ill.). The tubes will be capped and left at room temperature.After 15 minutes, the reactions are stopped by removing the Iodo-Beads.The ¹²⁵I-labeled peptides are then applied to 1 gm of C18 sorbent(Varian Bond ElutÒSPE column, Walnut Creek, Calif.) and washed with 10volumes of water containing 0.1% (w/v) cold iodine. Labeled peptides arethen be eluted with 3 volumes of 50% (v/v) acetonitrile water, and theradioactivity is determined using instruments known to those skilled inthe art, such as a MicroBeta TRILUX liquid Scintillation andluminescence counter (Wallac, Turku, Finland), and the radiolabeledpeptides are lyophilized.

Example 11 In Vitro Testing to Determine Efficacy of Laminin GlobularDomain-Derived Peptides as Aβ Amyloid Inhibitory Agents

The following are in vitro screening assays which are examples oftesting procedures to determine the efficacy of L- and D-lamininglobular domain derived peptides and analogs, as potential Aβ amyloidinhibitory agent.

Thioflavin T Fluorometry Assays:

Inhibition of Aβ fibril formation: Various peptides synthesized asoutlined above can be tested for potential Aβ amyloid inhibitoryactivity using in vitro assays. Thioflavin T fluorometry, which measuresthe amount of amyloid fibrils formed (LeVine III, Protein Sci.2:404–410, 1993; Amyloid: Int. J. Exp. Clin. Invest. 2:1–6, 1995; Naikiand Nakakuki, Lab. Invest., 74:374–383, 1996; Castillo et al, J.Neurochem. 69:2452–2465, 1997) can first be used to identify syntheticpeptides capable of inhibiting Aβ 1–40 amyloid fibril formation. Forthese studies, 25 μM of Aβ 1–40 (Bachem Inc) is incubated inmicrocentrifuge tubes at 37° C. for 1 week (in triplicate), eitheralone, or in the presence of 25 μM, 50 μM or 250 μM of parent peptidesor peptide-analogs (at Aβ:peptide molar ratios of 1:1; 1:2 and 1:10) in150 mM Tris HCl, 10 mM NaCl, pH 7.0 (TBS). 50 μl aliquots are taken foranalysis at 1 hour, 1 day, 3 days and 1 week and added to 1.2 ml of 100μM Thioflavin T and 50 mM NaPO4 (pH 6.0), respectively. Fluorescenceemission at 480 nm is measured on a Turner model 450 fluorometer at anexcitation wavelength of 450 nm. For each determination, the fluorometeris calibrated by zeroing in the presence of the Thioflavin T reagentalone, and by setting the 50 ng/ml riboflavin (Sigma) in the ThioflavinT reagent to 1800 fluorescence units. All fluorescence determinationsare based on these references and any fluorescence given off by peptidesin the presence of the Thioflavin T reagent is always subtracted fromall pertinent readings. Our experience indicates that Thioflavin T doesnot give off any false fluorescence in the presence of laminin-derivedpeptides, nor do these peptides cause any quenching problems. Previousstudies have also indicated that increasing concentrations of fibrillarAβ gives a proportional increase in fluorescence in the presence of 100μM Thioflavin T, ruling out the presence of any disproportionate innerfilter effects at this Thioflavin T concentration (Castillo et al J.Neurochem. 69:2452–2465, 1997).Disruption/Disassembly of Pre-formed Aβ Amyloid Fibrils: One can alsodetermine the dose-dependent ability of laminin globular domain-derivedpeptides (and peptide analogs) to disrupt/disassemble preformed Aβ 1–40and 1–42 fibrils. In these studies the peptides identified as inhibitorsof Aβ 1–40 amyloid fibril formation (as described above) are used. Forstudies involving fibrillar Aβ 1–40, 1 mg of Aβ 1–40 (Bachem Inc) isdissolved in 1.0 ml of double distilled water (1 mg/ml solution) andthen incubated at 37° C. for 1 week to cause abundant fibril formation.Aβ 1–42, which is already fibrillar, does not require pre-incubation at37° C. (as with Aβ 1–40), and is utilized immediately. For all of thesestudies, 25 μM of fibrillar amyloid (Aβ 1–40 or Aβ 1–42 is incubated inthe presence of 150 mM Tris-HCl, 10 mM NaCl (pH 7.0), in the presence orabsence of 25 μM, 50 μM, 125 μM and 250 μM of the various parentpeptides or peptide-analogs previously synthesized, giving approximateAβ:peptide molar ratios of 1:1, 1:2, 1:5, 1:10. All samples, includingcontrols (i.e. Aβ only, blank only or test-compound only) are tested intriplicate. Following an overnight, 3-day or 7-day incubation at 37° C.,50 μl aliquots are added to 1.2 ml of 100 μM Thioflavin T (Sigma) in 50mM NaPO4 (pH 6.0) for fluorometry readings as described above.Statistical Analysis: For the fibril formation/disruption assays asdescribed above, comparisons of Aβ 1–40 or Aβ 1–42 in the presence orabsence of peptides is based on paired Student's t tests with data shownas mean +/−S.E., or ANOVA, depending on the particular study.Significance is reported at the 95% (p<0.05), 99% (p<0.01), and 99.9%(p<0.001) confidence levels.Congo red Staining Assays: Aliquots (5 μl) from the incubation assays asdescribed above are also be analyzed by air-drying aliquots ongelatin-coated slides, followed by Congo red staining (Puchtler et al,J. Histochem. Cytochem. 10:355–364,1962). This technique has beeneffective in providing corroborating evidence of potential Aβ amyloidfibril inhibitors. A decrease in Congo red staining (i.e. red/greenbirefringence as viewed under polarized light) of fibrillar Aβ amyloidin the presence of peptides will confirm that a disruption/disassemblyof amyloid fibril architecture has taken place. Further analysis of themost potent peptides identified at the light microscopic level will alsoanalyzed by negative stain electron microscopy as described below.Negative Stain Electron Microscopy: Laminin globular domain-derivedpeptides (or peptide analogs) able to inhibit Aβ 1–40 fibril formation,and disrupter/disassemble pre-formed Aβ 1–42 fibrils, as determined byThioflavin T fluorometry and Congo red staining assays, (as describedabove), can be confirmed by negative stain electron microscopy. Forconfirmation of inhibition of Aβ 1–40 fibril formation, laminin globulardomain-derived peptides (or peptide analogs) are incubated with 50 μM offreshly solubilized Aβ 1–40 (Bachem) at Aβ:peptide molar ratios of 1:1,1:2 and 1:10, for increasing times (i.e. 0 hours, 1 day, 3 days and 7days) to observe any time-dependent and dose-dependent inhibition of Aβ1–40 fibril formation. Comparisons are made to Aβ 1–40 only. Negativelystained Aβ fibrils are prepared by floating pioloform, carbon-coatedgrids on peptide solutions (200 μg/ml of Aβ 1–40) in the presence ofabsence of various concentrations of laminin globular domain-derivedpeptides (as described above). To control for pH changes, peptides aredissolved in buffered solutions of 20 mM glycine (for pH 2 to 3 and pH 9to 10) or 20 mM Tris-HCl (for pH 6 to 8). After the grids are blottedand air-dried, the samples are stained with either 2% (w/v) uranylacetate or 1% (w/v) phosphotungstic acid and visualized, andphotographed, with instruments known to those skilled in the art, suchas a Phillips CM-10 electron microscope, using 80 kv acceleratingvoltage. The ability of peptides to disrupt the structure of amyloidfibrils can be qualitatively determined. In another study, negativestain electron microscopy can be utilized to confirm which lamininglobular domain-derived peptides (or peptide analogs) are effective indisruption/disassembly of pre-formed Aβ 1–42 fibrils. For these studies,50 μM of fibrillized Aβ 1–42 (prepared fresh) is incubated with lamininglobular domain-derived peptides (or peptide analogs) at Aβ:peptidemolar ratios of 1:1, 1:2 and 1:10 at 37° C. for 7 days. Aliquots aretaken at 0, 1, 3, and 7 days of incubation for analysis by negativestain electron microscopy as described above. Inhibitors/disruptors ofAβ fibrillogenesis are identified by their ability to form amorphousnon-fibrillar material. High magnification measurements (i.e. 100,000×)of Aβ amyloid fibrils (fibril diameter usually 7–10 nm) are compared tomaterials observed at 7 days following incubation with differentpeptides (as described above).

Further Aspects and Utilizations of the Invention

Laminin-Derived Polypeptides

One therapeutic application of the present invention is to uselaminin-derived polypeptides as potent inhibitors of Aβ amyloidformation, deposition, accumulation and/or persistence, in Alzheimer'sdisease, Down's syndrome and other amyloid disorders involving Aβfibrillogenesis.

The polypeptide referred to above may be a natural polypeptide, asynthetic polypeptide or a recombinant polypeptide. The polypeptides,derivatives or analogs referred to herein may be a) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue and such substituted amino acid residuemay or not be encoded by the genetic code, or b) in which one or more ofthe amino acid residues includes a substituent group, or c) one in whichthe mature polypeptide is fused with another compound, such as acompound used to increase the half-life of the polypeptide (for example,polylysine), or d) one in which the additional amino acids are fused tothe mature polypeptide, such as a leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such polypeptides, derivatives and analogs aredeemed to be within the scope of the invention.

Protein conformation is an essential component of protein-protein,protein-substrate, protein-agonist, protein-antagonist interactions.Changes in the component amino acids of protein sequences can result inchanges that have little or no effect on the resultant proteinconformation. Conversely, changes in the peptide sequences can haveeffects on the protein conformation resulting in reduced or increasedprotein-protein interactions. Such changes and their effects aregenerally disclosed in Proteins: Structures and Molecular Properties byThomas Creighton, W. H. Freeman and Company, New York, 1984 which ishereby incorporated by reference.

It will be appreciated by those skilled in the art that changes can bemade to the disclosed laminin polypeptides, derivatives or analogs, thatincrease, decrease or otherwise have no effect on the binding of lamininor fragments thereof to Aβ amyloid. In addition, it will be appreciatedby those skilled in the art that various post-translationalmodifications such as phosphorylation, glycosylation and the like, willalter the binding of laminin polypeptides, derivatives or analogs to Aβamyloid.

The polypeptides of the present invention include the polypeptidesdescribed herein, including but not limited to AG73 (SEQ ID NO:1), C-16(SEQ ID NO:2), A-13 (SEQ ID NO:3), HA3G47 (SEQ ID NO:4), HA3G58 (SEQ IDNO:5), HA3G67 (SEQ ID NO:6), HA3G74 (SEQ ID NO:7), HA3G76 (SEQ ID NO:8),HA3G79 (SEQ ID NO:9), HA3G83 (SEQ ID NO:10), A4G82 (SEQ ID NO:11), A5G15(SEQ ID NO:12), A5G56 (SEQ ID NO:13), A5G80 (SEQ ID NO: 14), A5G81 (SEQID NO:15), A5G82 (SEQ ID NO: 16), A5G84 (SEQ ID NO:17), A5G101 (SEQ IDNO:18), A5G109 (SEQ ID NO:19) (Sequence Group A), and fragments thereof,as well as polypeptides which preferably have at least a 70%, and morepreferably a 90% identity, to the polypeptides described above. “%Identity” as used herein for peptides means the same amino acids in thesame place. Thus a ten amino acid peptide that is identical to anotherten amino acid peptide, except for one amino acid, is 90% identical. Ifa ten amino acid peptide has the same ten amino acids in the same numberof each amino acid as another ten amino acid peptide, but two aminoacids are juxtaposed with each other, then the two amino acids have an80% identity with each other, and so forth.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture), or from a virus (such as with the use of phage displaytechniques known to those skilled in the art). Depending upon the hostemployed in a recombinant procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. Polypeptidesof the invention may also include an initial methionine amino acidresidue.

Chemical polypeptide synthesis is a rapidly evolving area in the art,and methods of solid phase polypeptide synthesis are well-described inthe following references, hereby entirely incorporated by reference(Merrifield, J. Amer. Chem. Soc. 85:2149–2154, 1963; Merrifield, Science232:341–347, 1986; Fields, Int. J. Polypeptide Prot. Res. 35, 161,1990).

Recombinant production of laminin polypeptides can be accomplishedaccording to known method steps. Standard reference works setting forththe general principles of recombinant DNA technology include Watson,Molecular Biology of the Gene, Volumes I and II, The Benjamin/CummingsPublishing Company Inc., publisher, Menlo Park, Calif. 1987; Ausubel etal, eds., Current Protocols in Molecular Biology, Wiley Interscience,publisher, New York, N.Y. 1987; 1992; and Sambrook et al, MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, publisher, Cold Spring Harbor, N.Y. 1989, the entirecontents of which references are herein incorporated by reference.

The polypeptides of the present invention may also be utilized asresearch reagents and materials for discovery of treatments anddiagnostics for human diseases.

Antibodies

Antibodies generated against the polypeptides corresponding to specificsequences recognizing the laminin fragments of the present inventionwhich bind Aβ can be obtained by direct injection of the polypeptidesinto an animal or by administering the polypeptides to an animal,preferably a nonhuman. The antibody so obtained will then bind thepolypeptides itself. In this manner, even a sequence encoding only afragment of the polypeptides can be used to generate antibodies bindingthe whole native polypeptides. Such antibodies can then be used toisolate the polypeptides from tissue expressing that polypeptide.Preferred embodiments include, but are not limited to, Sequence Group A,and fragments thereof, as well as polypeptides which have at least 70%identity and more preferably a 90% identity to the polypeptidesdescribed above.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies toantibodies specific for laminin-derived protein fragments orpolypeptides of the present invention.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.

A monoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. For preparation of monoclonal antibodies,any technique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohlerand Milstein, Nature 256:495–497, 1975), the trioma technique, the humanB-cell hybridoma technique (Kozbor et al, Immunology Today 4:72, 1983),and the EBV-hybridoma technique to produce human monoclonal antibodies(Cole et al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp.77–96, 1985). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, GILD and any subclass thereof.

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, which are primarily used to reduceimmunogenicity in application and to increase yields in production.Chimeric antibodies and methods for their production are known in theart (ex. Cabilly et al, Proc. Natl. Acad. Sci. U.S.A 81:3273–3277, 1984;Harlow and Lane: Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory 1988).

An anti-idiotypic antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An anti-idiotypic antibody can be prepared by immunizing ananimal of the same species and genetic type (e.g., mouse strain) as thesource of the monoclonal antibody with the monoclonal antibody to whichan anti-idiotypic antibody is being prepared. The immunized animal willrecognize and respond to the idiotypic determinants of the immunizingantibody by producing an antibody to these idiotypic determinants (theanti-idiotypic antibody). See, for example, U.S. Pat. No. 4,699,880,which is herein incorporated by reference.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab″)₂, whichare capable of binding antigen. Fab and F(ab″)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al, J. Nucl. Med. 24:316–325, 1983).

The antibodies or fragments of antibodies, useful in the presentinvention may be used to quantitatively or qualitatively detectlaminin-derived fragments in a sample or to detect presence of cellswhich express a laminin polypeptide of the present invention. This canbe accomplished by immunofluorescence techniques employing aflourescently labeled antibody coupled with light microscopic, flowcytometric or fluorometric detection.

One of the ways in which a laminin fragment antibody can be detectablylabeled is by linking the same to an enzyme and use in an enzymeimmunoassay (EIA). This enzyme, in turn, when later exposed to anappropriate substrate, will react with the substrate in such a manner asto produce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric, or by visual means. Enzymes which canbe used detectably label the antibody include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colometric methods which employ a chromogenic substrate for theenzyme. Detection can be accomplished by colometric methods which employa chromogenic substrate for the enzyme. Detection can also beaccomplished by visual comparison of the extent of enzymatic reaction ofa substrate with similarly prepared standards (see Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory 1988;Ausubel et al, eds., Current Protocols in Molecular Biology, WileyInterscience, N.Y. 1987, 1992).

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radiolabeling of the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work etal, North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, incorporated entirely by referenceherein. The radioactive isotope can be detected by such means as the useof a gamma-counter, a scintillation counter or by autoradiography.

It is also possible to label a laminin fragment polypeptide antibodywith a fluorescent compound. When the flourescently labeled antibody isexposed to light of the proper wave length, its presence can then bedetected due to fluorescence. Among the most commonly used fluorescentlabeling compounds are fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine, commercially available, e.g., from Molecular Probes, Inc.(Eugene, Oreg., U.S.A.).

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²EU, or other of the lanthanide series. These metalscan be attached to the antibody using such metal groups asdiethylenetriamine pentaacetic acid (EDTA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt, andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, lucifers and aequorin.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of a laminin fragmentof the present invention. In situ detection may be accomplished byremoving a histological specimen from a patient, and providing thelabeled antibody of the present invention to such a specimen. Theantibody (or fragment) is preferably provided by applying or byoverlaying the labeled antibody (or fragment) to a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of a laminin fragment polypeptide but also itsdistribution on the examined tissue. Using the present invention, thoseof ordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Antibodies against laminin fragments and/or laminin-derived polypeptideswhich interact with Aβ or other amyloid proteins, or derivatives thereofare also disclosed herein. These antibodies can be used for a number ofimportant diagnostic and/or therapeutic applications as describedherein. In one aspect of the invention, polyclonal and/or monoclonalantibodies made against laminin fragments and/or laminin-derivedpolypeptides which bind Aβ or other amyloid proteins, may be utilizedfor Western blot analysis (using standard Western blotting techniquesknowledgeable to those skilled in the art) to detect the presence ofamyloid protein-binding laminin fragments or amyloid protein-bindinglaminin polypeptides in human tissues and in tissues of other species.Western blot analysis can also be used to determine the apparent size ofeach amyloid protein-binding laminin fragment. In addition, Westernblotting following by scanning densitometry (known to those skilled inthe art) can be used to quantitate and compare levels of each of thelaminin fragments or polypeptides in tissue samples, biological fluidsor biopsies obtained from individuals with specific diseases (such asthe amyloid diseases) in comparison to tissue samples, biological fluidsor biopsies obtained from normal individuals or controls. Biologicalfluids, include, but are not limited to, blood, plasma, serum,cerebrospinal fluid, sputum, saliva, urine and stool.

In yet another aspect of the invention, polyclonal and/or monoclonalantibodies made against laminin fragments and/or laminin-derivedpeptides which bind Aβ or other amyloid proteins, can be utilized forimmunoprecipitation studies (using standard immunoprecipitationtechniques known to one skilled in the art) to detect laminin fragmentsand/or laminin-derived peptides which bind Aβ or other amyloid proteins,in tissues, cells and/or biological fluids. Use of the laminin fragmentand/or laminin-derived peptide antibodies for immunoprecipitationstudies can also be quantitated to determine relative levels of lamininfragments and/or laminin-derived peptides which interact with Aβ orother amyloid proteins, in tissues, cells and/or biological fluids.Quantitative immunoprecipitation can be used to compare levels oflaminin fragments and/or laminin amyloid protein-binding peptides intissue samples, biological fluids or biopsies obtained from individualswith specific diseases (such as the amyloid diseases) in comparison totissue samples, biological fluids or biopsies obtained from normalindividuals or controls.

Therapeutic Applications

Yet another aspect of the present invention is to make use of lamininfragments and/or laminin-derived polypeptides as amyloid inhibitorytherapeutic agents. The laminin-derived peptide sequences or fragmentscan be synthesized utilizing standard techniques (i.e. using anautomated synthesizer). Laminin fragments and/or laminin-derivedpolypeptides which bind Aβ or other amyloid proteins, can be used aspotential blocking therapeutics for the interaction of laminin in anumber of biological processes and diseases (such as in the amyloiddiseases described above). In a preferred embodiment, specificlaminin-derived polypeptides may be used to aid in the inhibition of Aβamyloid formation, deposition, accumulation, and/or persistence in agiven patient. Likewise, in another preferred embodiment anti-idiotypicantibodies made against laminin fragments and/or laminin-derivedpolypeptides (as described above) may be given to a human patient aspotential blocking antibodies to disrupt continued Aβ amyloid formation,deposition, accumulation and/or persistence in the given patient.

Preparations of laminin-derived polypeptides for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, and emulsions, which may contain axillary agents orexcipients which are known in the art. Pharmaceutical compositions suchas tablets, pills, tablets, caplets, soft and hard gelatin capsules,lozenges, sachets, cachets, vegicaps, liquid drops, elixers,suspensions, emulsions, solutions, syrups, tea bags, aerosols (as asolid or in a liquid medium), suppositories, sterile injectablesolutions, sterile packaged powders, can be prepared according toroutine methods and are known in the art.

In yet another aspect of the invention, laminin fragments and/orlaminin-derived polypeptides may be used as an effective therapy toblock Aβ amyloid formation, deposition, accumulation and/or persistenceas observed in the amyloid diseases. For example, the invention includesa pharmaceutical composition for use in the treatment of amyloidosescomprising a pharmaceutically effective amount of a laminin fragmentand/or laminin-derived polypeptide anti-idiotypic antibody and apharmaceutically acceptable carrier. The compositions may contain thelaminin fragments and/or laminin-derived polypeptide anti-idiotypicantibody, either unmodified, conjugated to a potentially therapeuticcompound, conjugated to a second protein or protein portion or in arecombinant form (i.e. chimeric or bispecific laminin fragment and/orlaminin polypeptide antibody). The compositions may additionally includeother antibodies or conjugates. The antibody compositions of theinvention can be administered using conventional modes of administrationincluding, but not limited to, topical, intravenous, intra-arterial,intraperitoneal, oral, intralymphatic, intramuscular or intralumbar.Intravenous administration is preferred. The compositions of theinvention can be a variety of dosage forms, with the preferred formdepending upon the mode of administration and the therapeuticapplication. Optimal dosage and modes of administration for anindividual patient can readily be determined by conventional protocols.

Laminin-derived protein fragments, and laminin-derived polypeptides, orantibodies of the present invention may be administered by any meansthat achieve their intended purpose, for example, to treat laminininvolved pathologies, such as Alzheimer's disease and other amyloiddiseases, or other related pathologies, using a laminin-derivedpolypeptide described herein, in the form of a pharmaceuticalcomposition.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, transdermal or buccalroutes. Alternatively, or concurrently, administration may be by theoral route. Parenteral administration can be by bolus injection or bygradual perfusion over time.

A preferred mode of using a laminin-derived polypeptide, or antibodypharmaceutical composition of the present invention is by oraladministration or intravenous application.

A typical regimen for preventing, suppressing or treatinglaminin-involved pathologies, such as Alzheimer's disease amyloidosis,comprises administration of an effective amount of laminin-derivedpolypeptides, administered over a period of one or several days, up toand including between one week and about 24 months.

It is understood that the dosage of the laminin-derived polypeptides ofthe present invention administered in vivo or in vitro will be dependentupon the age, sex, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. The most preferred dosage will be tailored to theindividual subject, as is understood and determinable by one of skill inthe art, without undue experimentation.

The total dose required for each treatment may be administered bymultiple doses or in a single dose. A laminin-derived polypeptide may beadministered alone or in conjunction with other therapeutics directed tolaminin-involved pathologies, such as Alzheimer's disease or other Aβamyloid diseases, as described herein.

Effective amounts of a laminin-derived polypeptide or composition, whichmay also include a laminin-fragment derived antibody, are about 0.01 μμgto about 100 mg/kg body weight, and preferably from about 10 μg to about50 mg/kg body weight, such as 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9., 1,2, 5, 10, 20, 25, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 mg/kg.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which may containaxillary agents or excipients which are known in the art. Pharmaceuticalcompositions comprising at least one laminin-derived polypeptide, suchas 1–10 or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 laminin-derivedpolypeptides, of the present invention may include all compositionswherein the laminin-derived polypeptide is contained in an amounteffective to achieve its intended purpose. In addition to at least onelaminin-derived polypeptide, a pharmaceutical composition may containsuitable pharmaceutically acceptable carriers, such as excipients,carriers and/or axillaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions comprising at least one laminin-derivedpolypeptide or antibody may also include suitable solutions foradministration intravenously, subcutaneously, dermally, orally,mucosally, rectally or may by injection or orally, and contain fromabout 0.01 to 99 percent, preferably about 20 to 75 percent of activecomponent (i.e. polypeptide or antibody) together with the excipient.Pharmaceutical compositions for oral administration include pills,tablets, caplets, soft and hard gelatin capsules, lozenges, sachets,cachets, vegicaps, liquid drops, elixers, suspensions, emulsions,solutions, and syrups.

The laminin-derived protein fragments, and laminin-derived polypeptidesfor Alzheimer's disease and other central nervous system Aβ amyloidosesmay be optimized to cross the blood-brain barrier. Methods ofintroductions include but are not limited to systemic administration,parenteral administration i.e., via an intraperitoneal, intravenous,perioral, subcutaneous, intramuscular, intraarterial, intradermal,intramuscular, intranasal, epidural and oral routes. In a preferredembodiment, laminin-derived protein fragments, and laminin-derivedpolypeptides may be directly administered to the cerebrospinal fluid byintraventricular injection. In a specific embodiment, it may bedesirable to administer laminin-derived protein fragments, andlaminin-derived polypeptides locally to the area or tissue in need oftreatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, byinjection, by infusion using a cannulae with osmotic pump, by means of acatheter, by means of a suppository, or by means of an implant.

In yet another embodiment laminin-derived protein fragments, andlaminin-derived polypeptides may be delivered in a controlled releasesystem, such as an osmotic pump. In yet another embodiment, a controlledrelease system can be placed in proximity to the therapeutic target,i.e. the brain, thus requiring only a fraction of the systemic dose.

In yet another aspect of the present invention, peptidomimetic compoundsmodeled from laminin fragments and/or laminin-derived polypeptidesidentified as binding Aβ or other amyloid proteins, may serve as potentinhibitors of amyloid formation, deposition, accumulation and/orpersistence in Alzheimer's disease and other Aβ amyloidoses.Peptidomimetic modeling is implemented by standard procedures known tothose skilled in the art.

In yet another aspect of the present invention, compounds that mimic the3-dimensional Aβ binding site on laminin using computer modeling, mayserve as potent inhibitors of Aβ amyloid formation, deposition,accumulation and/or persistence in Alzheimer's disease and other Aβamyloidoses. Design and production of such compounds using computermodeling technologies is implemented by standard procedures known tothose skilled in the art.

Recombinant DNA technology, including human gene therapy, has directapplicability to the laminin polypeptides, of this invention. Oneskilled in the art can take the peptide sequences disclosed herein andcreate corresponding nucleotide sequences that code for thecorresponding peptide sequences. These sequences can be cloned intovectors such as retroviral vectors, and the like. These vectors can, inturn, be transfected into human cells such as hepatocytes orfibroblasts, and the like. Such transfected cells can be introduced intohumans to treat amyloid diseases. Alternatively, the genes can beintroduced into the patients directly. The basic techniques ofrecombinant DNA technology are known to those of ordinary skill in theart and are disclosed in Recombinant DNA Second Edition, Watson, et al.,W.H. Freeman and Company, New York, 1992, which is hereby incorporatedby reference.

Diagnostic Applications

Another aspect of the invention is to provide polyclonal and/ormonoclonal antibodies against laminin fragments and/or laminin-derivedpolypeptides which bind Aβ or other amyloid proteins, which is utilizedto specifically detect laminin fragments and/or laminin-derived peptidesin human tissues and/or biological fluids. In one preferred embodiment,polyclonal or monoclonal antibodies made against a peptide portion orfragment of laminin, can be used to detect and quantify lamininfragments and/or laminin-derived polypeptides in human tissues and/orbiological fluids. Polyclonal and/or monoclonal peptide antibodies canalso be utilized to specifically detect laminin fragments and/orlaminin-derived polypeptides in human tissues and/or biological fluids.In a preferred embodiment, a polyclonal or monoclonal antibody madespecifically against a peptide portion or fragment of laminin fragmentsor polypeptides which bind Aβ (as described herein), can be used todetect and quantify these laminin fragments or polypeptides in humantissues and/or biological fluids. Other preferred embodiments include,but are not limited to, making polyclonal or monoclonal antibodies madespecifically against a peptide portion or fragment of any of thepeptides of Sequence Group A, as well as polypeptides which have atleast 70% identity and more preferably a 90% identity to thepolypeptides described above. For detection of laminin fragments and/orlaminin-derived polypeptides described above in human tissues, cells,and/or in cell culture, the polyclonal and/or monoclonal antibodies canbe utilized using standard immunohistochemical and immunocytochemicaltechniques, known to one skilled in the art.

For detection and quantitation of laminin fragments and/orlaminin-derived polypeptides in biological fluids, includingcerebrospinal fluid, blood, plasma, serum, urine, sputum, and/or stool,various types of ELISA assays can be utilized, known to one skilled inthe art. An antibody molecule of the present invention may be adaptedfor utilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier, and a quantity of detectable labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

In a preferred embodiment, a “sandwich” type of ELISA can be used. Usingthis preferred method a pilot study is first implemented to determinethe quantity of binding of each laminin-fragment or polypeptidemonoclonal antibody to microtiter wells. Once this is determined,aliquots (usually in 40 μl of TBS; pH 7.4) of the specificlaminin-fragment or laminin polypeptide antibody are allowed to bindovernight to microtiter wells (Maxisorb C plate from Nunc) at 4° C. Aseries of blank wells not containing any laminin-fragment or lamininpolypeptide specific monoclonal antibody are also utilized as controls.The next day, non-bound monoclonal antibody is shaken off the microtiterwells. All of the microtiter wells (including the blank wells) are thenblocked by incubating for 2 hours with 300 μl of Tris-buffered salinecontaining 0.05% Tween-20 (TTBS) plus 2% bovine serum albumin, followedby 5 rinses with TTBS. 200 μl of cerebrospinal fluid, blood, plasma,serum, urine, sputum, and/or stool and/or any other type of biologicalsample is then diluted (to be determined empirically) in TTBS containing2% bovine serum albumin and placed in wells (in triplicate) containingbound laminin-fragment or laminin-polypeptide antibody (or blank) andincubated for 2 hours at room temperature. The wells are then washed 5times with TTBS. A second biotinylated-monoclonal antibody against thesame laminin-derived fragment or laminin polypeptide (but which isagainst a different epitope) is then added to each well (usually in 40μl of TBS; pH 7.4) and allowed to bind for 2 hours at room temperatureto any laminin-fragment or laminin polypeptide captured by the firstantibody. Following incubation, the wells are washed 5 times with TTBS.Bound materials are then detected by incubating with 100 μl ofperoxidase-avidin complex (1:250 dilution in TTBS with 0.1% BSA) for 1hour on a rotary shaker. After 5 washes with TTBS, a substrate solution(100 μl, OPD-Sigma Fast from Sigma Chemical Co., St. Louis, Mo., USA) isadded and allowed to develop significant color (usually 8–10 minutes).The reaction is stopped with 50 μl of 4N sulfuric acid and read on astandard spectrophotometer at 490 nm. This ELISA can be utilized todetermine differences in specific laminin fragments or polypeptides(and/or Aβ-binding laminin fragments or polypeptides) in biologicalfluids which can serve as a diagnostic marker to follow the progressionin a live patient during the progression of disease (i.e. monitoring ofAβ amyloid disease as an example). In addition, quantitative changes inlaminin fragments or laminin polypeptides can also serve as a prognosticindicator monitoring how a live patient will respond to treatment whichtargets an Aβ amyloid disease such as Alzheimer's disease. Such assayscan be provided in a kit form.

A competition assay may also be employed wherein antibodies specific tolaminin fragments and/or laminin-derived polypeptides are attached to asolid support and labeled laminin fragments and/or laminin-derivedpolypeptides and a sample derived from a host are passed over the solidsupport and the amount of label detected attached to the solid supportcan be correlated to the quantity of laminin fragments and/orlaminin-derived polypeptides in the sample. This standard technique isknown to one skilled in the art.

Another object of the present invention is to use laminin fragmentsand/or laminin-derived polypeptides, in conjunction with lamininfragment and/or laminin-derived peptide antibodies, in an ELISA assay todetect potential laminin fragment and/or laminin-derived peptideautoantibodies in human biological fluids. Such a diagnostic assay maybe produced in a kit form. In a preferred embodiment, peptidescontaining the sequences of laminin-derived fragments andlaminin-derived polypeptides as in Sequence Group A, as well aspolypeptides which have at least 70% identity and more preferably a 90%identity to the polypeptides described above, will be used to initiallybind to microtiter wells in an ELISA plate.

A pilot study is first implemented to determine the quantity of bindingof each laminin fragment or polypeptide to microtiter wells. Once thisis determined, aliquots (usually 1–2 μg in 40 μl of TBS; pH 7.4) ofspecific laminin fragment polypeptides (as described herein) are allowedto bind overnight to microtiter wells (Maxisorb C plate from Nunc) at 4°C. All the microtiter wells (including blank wells without the lamininfragment polypeptides) are blocked by incubating for 2 hours with 300 μlof Tris-buffered saline (pH 7.4) with 0.05% Tween-20 (TTBS), containing2% albumin. This is followed by 5 rinses with TTBS. The patients'biological fluids (i.e., cerebrospinal fluid, blood, plasma, serum,sputum, urine, and/or stool) are then utilized and 200 μl are diluted(to be determined empirically) with TTBS containing 2% bovine serumalbumin, and placed in microtiter wells (in triplicate) containing aspecific laminin fragment polypeptide or blank wells (which do notcontain peptide), and are incubated at 1.5 hours at room temperature.

Any autoantibodies present in the biological fluids against the lamininfragment or polypeptide will bind to the substrate bound lamininfragment polypeptide (or fragments thereof). The wells are then rinsedby washing 5 times with TTBS. 100 μl of biotinylated polyclonal goatanti-human IgG's (Sigma Chemical company, St. Louis, Mo., USA), diluted1:500 in TTBS with 0.1% bovine serum albumin, is then aliquoted intoeach well. Bound materials are detected by incubating with 100 μl ofperoxidase-avidin complex (1:250 dilution in TTBS with 0.1% bovine serumalbumin) for 1 hour on a rotary shaker. Following 5 washes with TTBS,substrate solution (100 μl, OPD-Sigma Fast from Sigma Chemical Company,St. Louis, Mo., USA) is added and allowed to develop significant color(usually 8–10 minutes). The reaction is stopped with 50 μl of 4Nsulfuric acid added to each well and read on a standardspectrophotometer at 490 nm.

This assay system can be utilized to not only detect the presence ofautoantibodies against laminin fragments or polypeptides in biologicalfluids, but also to monitor the progression of disease by followingelevation or diminution of laminin fragment or polypeptide autoantibodylevels. It is believed that patients demonstrating excessive lamininfragment or polypeptide formation, deposition, accumulation and/orpersistence as may be observed in the Aβ amyloid diseases, will alsocarry autoantibodies against the laminin fragments or lamininpolypeptides in their biological fluids. Various ELISA assay systems,knowledgeable to those skilled in the art, can be used to accuratelymonitor the degree of laminin fragments or polypeptides in biologicalfluids as a potential diagnostic indicator and prognostic marker forpatients during the progression of disease (i.e. monitoring of an Aβamyloid disease for example). Such assays can be provided in a kit form.In addition, quantitative changes in laminin fragment or polypeptideautoantibody levels can also serve as a prognostic indicator monitoringhow a live patient will respond to treatment which targets an Aβ amyloiddisease.

Other diagnostic methods utilizing the invention include diagnosticassays for measuring altered levels of laminin fragments and/orlaminin-derived polypeptides in various tissues compared to normalcontrol tissue samples. Assays used to detect levels of lamininfragments and/or laminin-derived polypeptides in a sample derived from ahost are well-known to those skilled in the art and includedradioimmunoassays, competitive-binding assays, Western blot analysis andpreferably ELISA assays (as described above).

Yet another aspect of the present invention is to use the antibodiesrecognizing laminin fragments and/or laminin-derived polypeptides forlabelings, for example, with a radionucleotide, for radioimaging orradioguided surgery, for in vivo diagnosis, and/or for in vitrodiagnosis. In one preferred embodiment, radiolabeled peptides orantibodies made (by one skilled in the art) against laminin fragmentsand/or laminin-derived polypeptides may be used as minimally invasivetechniques to locate laminin fragments and/or laminin-derivedpolypeptides, and concurrent Aβ amyloid deposits in a living patient.These same imaging techniques can then be used at regular intervals(i.e. every 6 months) to monitor the progression of the Aβ amyloiddisease by following the specific levels of laminin fragments and/orlaminin-derived polypeptides.

Yet another aspect of the present invention is to provide a method whichcan evaluate a compound's ability to alter (diminish or eliminate) theaffinity of Aβ (as described herein) or amyloid precursor protein,to'laminin-derived fragments or laminin-derived polypeptides. Byproviding a method of identifying compounds which affect the binding ofAβ amyloid protein, or amyloid precursor protein to such laminin-derivedfragments or polypeptides, the present invention is also useful inidentifying compounds which can prevent or impair such bindinginteractions. Thus, compounds can be identified which specificallyaffect an event linked with Aβ amyloid formation, amyloid deposition,and/or amyloid persistence condition associated with Alzheimer's diseaseand other Aβ amyloid diseases as described herein.

According to one aspect of the invention, to identify for compoundswhich allow the interaction of Aβ†amyloid proteins or precursor proteinsto laminin-derived fragments or laminin polypeptides, either Aβ†amyloidor laminin fragments or polypeptides are immobilized, and the other ofthe two is maintained as a free entity. The free entity is contactedwith the immobilized entity in the presence of a test compound for aperiod of time sufficient to allow binding of the free entity to theimmobilized entity, after which the unbound free entity is removed.Using antibodies that recognize the free entity, or other means todetect the presence of bound components, the amount of free entity boundto immobilized entity can be measured. By performing this assay in thepresence of a series of known concentrations of test compound and, as acontrol, the complete absence of test compound, the effectiveness of thetest compound to allow binding of free entity to immobilized entity canbe determined and a quantitative determination of the effect of the testcompound on the affinity of free entity to immobilized entity can bemade. By comparing the binding affinity of the Aβ amyloid-lamininfragment or polypeptide complex in the presence of a test compound tothe binding affinity of the amyloid-laminin fragment or polypeptidecomplex in the absence of a test compound, the ability of the testcompound to modulate the binding can be determined. In the case in whichthe Aβ amyloid is immobilized, it is contacted with free laminin-derivedfragments or polypeptides, in the presence of a series of concentrationsof test compound. As a control, immobilized Aβ amyloid is contacted withfree laminin-derived polypeptides, or fragments thereof in the absenceof the test compound. Using a series of concentrations oflaminin-derived polypeptides, the dissociation constant (K_(d)) or otherindicators of binding affinity of amyloid-laminin fragment orpolypeptide binding can be determined. In the assay, after thelaminin-derived polypeptides or fragments thereof are placed in contactwith the immobilized Aβ amyloid for a sufficient time to allow binding,the unbound laminin polypeptides are removed. Subsequently, the level oflaminin fragment or polypeptide-Aβ amyloid binding can be observed. Onemethod uses laminin-derived fragment or polypeptide antibodies, asdescribed in the invention, to detect the amount of specific lamininfragments or polypeptides bound to the Aβ amyloid or the amount of freelaminin fragments remaining in solution. This information is used todetermine first qualitatively whether or not the test compound can allowcontinued binding between laminin-derived fragments or polypeptides andAβ amyloid. Secondly, the data collected from assays performed using aseries of test compounds at various concentrations, can be used tomeasure quantitatively the binding affinity of the laminin fragment orpolypeptide-Aβ amyloid complex and thereby determine the effect of thetest compound on the affinity between laminin fragments or polypeptidesand Aβ amyloid. Using this information, compounds can be identifiedwhich do not modulate the binding of specific laminin fragments orpolypeptides to amyloid and thereby allow the laminin-fragments orpolypeptides to reduce the Aβ amyloid formation, deposition,accumulation and/or persistence, and the subsequent development andpersistence of Aβ amyloidosis.

Therefore a kit for practicing a method for identifying compounds usefulwhich do not alter laminin-derived fragments or laminin-derivedpolypeptides to an immobilized Aβ amyloid protein, said kit comprisinga) a first container having Aβ amyloid protein immobilized upon theinner surface, b) a second container which contains laminin-derivedfragments or laminin-derived polypeptides dissolved in solution, c) athird container which contains antibodies specific for saidlaminin-derived fragments or laminin-derived polypeptides, saidantibodies dissolved in solution, and d) a fourth container whichcontains labeled antibodies specific for laminin-derived fragments orlaminin-derived polypeptides, said antibodies dissolved in solution.

Amyloid Enhancing Agents

The use of specific laminin-derived peptides for the formation, andenhancement of formation, of amyloid maltese-cross plaque-like deposits,or Aβ amyloid enhancing agents is also disclosed herein. Theseamyloid-enhancing compounds are capable of increasing the rate of Aβamyloid formation both in vivo and in vitro. Thus methods of inducing Aβamyloid formation, which resembles the congophilic maltese-cross amyloidplaques of Alzheimer's disease and Down's syndrome are also disclosedherein.

In one aspect, the invention features a method of forming amyloidfibrils from an amyloidogenic peptide derived from laminin. In preferredembodiments, the amyloid compound is a polypeptide selected from thegroup consisting of Sequence Group C. The features incubating alaminin-derived polypeptide as described above at 37° C., either in theabsence of presence of Aβ (1–40 or 1–42). Amyloid plaque-like formationis observed following the staining with Congo red, and viewing underpolarized light. A maltese-cross amyloid-like plaque is surprisinglyformed.

In an alternate aspect, a method of increasing amyloid deposition in amammal is disclosed. The method comprises administering to the mammal aneffective amount of an amyloid-enhancing compound. In preferredembodiments, the amyloid enhancing compounds is a polypeptide selectedfrom the group consisting of Sequence Group C. The polypeptides of theinvention can be administered to an animal by a route which is effectivefor enhancing amyloid deposition. Suitable routes of administrationinclude subcutaneous, intravenous and intraperitoneal injection, andoral administration. The compounds can be administered with apharmaceutically acceptable vehicle.

In still another aspect of the invention features a method for screeningfor agents useful for treating Aβ amyloidosis. The method comprisesproviding a reaction mixture which includes a solution of anamyloidogenic peptide (such as Aβ 1–40 or Aβ 1–42), an amyloid-enhancingcompound (such as the amyloid-enhancing laminin-derived polypeptidesdescribed above, and an agent potentially useful for treating Aβamyloidosis, under conditions such that, in the absence of the agentpotentially useful for treating Aβ amyloidosis, amyloid fibrils oramyloid plaque-like structures would form, and observing formation orabsence of amyloid fibrils or amyloid plaque-like structures.

With regard to systems and components above referred to, but nototherwise specified or described in detail herein, the workings andspecifications of such systems and components and the manner in whichthey may be made or assembled or used, both cooperatively with eachother and with the other elements of the invention described herein toeffect the purposes herein disclosed, are all believed to be well withinthe knowledge of those skilled in the art. No concerted attempt torepeat here what is generally known to the artisan has therefore beenmade.

In compliance with the statute, the invention has been described inlanguage more or less specific as to structural features. It is to beunderstood, however, that the invention is not limited to the specificfeatures shown, since the means and construction shown comprisepreferred forms of putting the invention into effect. The invention is,therefore, claimed in any of its forms or modifications within thelegitimate and valid scope of the appended claims, appropriatelyinterpreted in accordance with the doctrine of equivalents.

1. A pharmaceutical composition comprising a peptide HA3G76 consistingof Tyr-Leu-Ser-Lys-Gly-Arg-Leu-Val-Phe-Ala-Leu-Gly (SEQ ID NO:8).
 2. Thepharmaceutical composition of claim 1 wherein any individual amino acidwithin the peptide may be either L- or D-amino acid.
 3. Thepharmaceutical composition of claim 1 further comprising apharmaceutically acceptable carrier, diluent or excipient.
 4. Thepharmaceutical composition of claim 1 wherein the individual amino acidswithin the peptide are D-amino acids.